Process for the Fermentative Preparation of Organic Chemical Compounds Using Coryneform Bacteria in which the SugR Gene is Present in Attenuated Form

ABSTRACT

The invention relates to a recombinant coryneform bacterium which secretes an organic chemical compound and in which the sugR gene which codes for a polypeptide having the activity of an SugR regulator has been attenuated. The invention further relates to a processes for using this bacterium for the fermentative preparation of organic chemical compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 12/232,610,filed on Sep. 19, 2008, which claims the benefit of U.S. provisionalapplication 60/960,375 filed on Sep. 27, 2007 and to U.S. provisionalapplication 60/996,706 filed on Nov. 30, 2007. These prior applicationsare hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to coryneform bacteria which secrete organicchemical compounds and in which the sugR gene is present in attenuatedform. It is also directed to processes for the fermentative preparationof organic chemical compounds, where the medium comprises as a carbonsource a mixture of one or more of the sugars selected from the group ofglucose, fructose and sucrose, and acetic acid.

BACKGROUND OF THE INVENTION

Organic chemical compounds, particularly amino acids, vitamins, hydroxyacids, keto acids, nucleosides and nucleotides, are used in humanmedicine, in the pharmaceutical industry, in cosmetics, in the foodindustry and in livestock nutrition. Many of these compounds areprepared by the fermentation of strains of coryneform bacteria,especially Corynebacterium glutamicum. These fermentation procedures arecontinually being improved by measures relating to: fermentationtechnology (e.g., changes in stirring or the supply of oxygen); thecomposition of the nutrient medium (e.g., the sugar concentration duringthe fermentation); the working up of the product formed (e.g., by ionexchange chromatography); or the intrinsic output properties of themicroorganism itself.

Methods used for improving the output properties of bacteria may involvemutagenesis, or changes in the selection and choice of mutants. Forexample, strains may be developed that produce the organic chemicalcompound and that are resistant to antimetabolites. Methods ofrecombinant DNA technology have been employed for some years forimproving of L-amino acid-producing strains of Corynebacteriumglutamicum. A summary of various aspects of the genetics, metabolism andbiotechnology of Corynebacterium glutamicum may be found in Pühler((chief ed.) (J. Biotechnol. 104 (1-3): 1-338 (2003)) and Eggeling, etal. ((editors) Handbook of Corynebacterium Glutamicum, CRC Press, Taylor& Francis Group, Boca Raton (2005)).

Nucleotide sequences of the genes or open reading frames (ORF) ofCorynebacterium glutamicum ATCC13032 form part of the prior art and canbe determined, inter alia, from the genomic sequence published byKalinowski et al. (J. Biotechnol. 104:5-25 (2003), Access No.NC_(—)006958)).

Nucleotide sequences of the genes or open reading frames (ORF) ofCorynebacterium glutamicum R also form part of the prior art and can bedetermined, inter alia, from the genomic sequence published by Yukawa etal. (Microbiol. 153(4):1042-1058 (2007)), Accession No. NC_(—)009342).

The nucleotide sequences of the genes or open reading frames (ORF) ofCorynebacterium efficiens likewise form part of the art and can bedetermined, inter alia, from the genomic sequence published by Nishio,et al. (Genome Res. 13:1572-1579 (2003), Accession No. NC_(—)004369).

In addition, numerous nucleotide sequences of Corynebacteriumthermoaminogenes are known.

Despite this plethora of sequence data, there are numerous ORFs forwhich no clear function has been assignable to date. Glucose or sucroseis mostly used as carbon source for the fermentative preparation oforganic chemical compounds with the aid of coryneform bacteria. There isa continuous search for alternative suitable raw materials or rawmaterial mixtures.

It is known that Corynebacterium glutamicum can utilize acetic acid as acarbon source. Investigations on the utilization of mixtures of carbonsources, for example mixtures comprising glucose and one or more of thecompounds selected from the group of acetic acid, lactate and fructoseare described by Cocaign et al. (Appl. Microbiol. Biotechnol. 40:526-530(1993)), Dominguez, et al. (Appl. Microbiol. Biotechnol. 47(5):600-603(1997)), Dominguez, et al. (Eur. J. Biochem. 254(1):96-102. (1998)) andWendisch, et al. (J. Bacteriol. 182(11):3088-96 (2000)).

U.S. Pat. No. 4,368,266 describes a process for preparing L-glutamicacid by using acetic acid as a carbon source with the aid of coryneformbacteria having a defect in isocitrate lyase.

U.S. Pat. No. 4,728,610 describes a process for preparing L-glutamicacid by using carbohydrates and acetic acid as carbon source with theaid of coryneform bacteria.

Wendisch et al. (J. Bacteriol. 182(11): 3088-96 (2000)) observed areduced glucose uptake when acetic acid and glucose were metabolizedtogether.

OBJECT OF THE INVENTION

The object addressed by the inventors was to provide novel coryneformbacteria able to utilize mixtures of carbon sources comprising one ormore of the sugars selected from the group consisting of glucose,fructose and sucrose and acetic acid for the effective formation andenrichment of organic chemical compounds. A further object directlyconnected thereto was to provide an improved process for thefermentative preparation of organic chemical compounds, especially aminoacids, vitamins, α-keto acids, nucleosides and nucleotides, with the aidof such coryneform bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of the plasmid pK19mobsacB_deltasugR.

DESCRIPTION OF THE INVENTION

The invention relates to recombinant coryneform bacteria which secreteorganic chemical compounds and in which the sugR gene, which codes forthe SugR regulator, is present in attenuated form These bacteria havethe ability to utilize one or more of the sugars selected from the groupof glucose, fructose and sucrose, and acetic acid as their carbonsource.

Coryneform bacteria, preferably of the genus Corynebacterium, are usedto prepare the bacteria of the invention. Strains derived from thefollowing species are particularly preferred:

-   -   Corynebacterium efficiens such as, for example, the type strain        DSM44549,    -   Corynebacterium glutamicum such as, for example, the type strain        ATCC13032 or the strain R, and    -   Corynebacterium ammoniagenes such as, for example, the strain        ATCC6871, with very particular preference for the species        Corynebacterium glutamicum.

Some representatives of the species Corynebacterium glutamicum are knownin the prior art under other species names. These include for example:

Corynebacterium acetoacidophilum ATCC13870

Corynebacterium lilium DSM20137

Corynebacterium melassecola ATCC 17965

Brevibacterium flavum ATCC 14067

Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaricatum ATCC 14020.

The term “Micrococcus glutamicus” for Corynebacterium glutamicum haslikewise been used.

Some representatives of the species Corynebacterium efficiens have alsobeen referred to in the prior art as Corynebacterium thermoaminogenessuch as, for example, the strain FERM BP-1539.

The coryneform bacteria employed for attenuation measures have, interalia, the ability to utilize the sugars glucose, fructose, sucrose andacetic acid singly or together as a carbon source.

The strains of coryneform bacteria (starting strains) employed forattenuation measures preferably already have the capability of producingorganic chemical compound(s) that are enriched in the cell or secretedinto nutrient medium. The term “produce” refers to processes entailingeither enrichment or secretion. In particular, the strains of coryneformbacteria employed have the ability to enrich or to accumulate ≧(atleast) 0.25 g/l, ≧0.5 g/l, ≧1.0 g/l, ≧1.5 g/l, ≧2.0 g/l, ≧4 g/l or ≧10g/l of the desired compound in ≦(at most) 120 hours, ≦96 hours, ≦48hours, ≦36 hours, ≦24 hours or ≦12 hours in the cell or in the nutrientmedium. The starting strains are preferably strains which have beenprepared by mutagenesis and selection, by recombinant DNA techniques orby a combination of the two methods.

Bacteria according to the invention can also be obtained by firstattenuating the sugR gene in a wild strain such as, for example,ATCC13032, and then genetically engineering the bacteria to produce thedesired organic chemical compound(s).

It is further advantageous to carry out the attenuation measures on thesugR gene in strains which require acetic acid as a supplement in thenutrient medium. For example, EP 1767 616 A1 describes strains withattenuated pyruvate dehydrogenase which require acetic acid as a sourcein the medium to prepare L-amino acids.

The term “organic chemical compound” includes amino acids, vitamins,hydroxy acids, keto acids, nucleosides and nucleotides.

The term “amino acids” includes D-amino acids and L-amino acids.Preferred L-amino acids are the proteinogenic amino acids, L-ornithineand L-homoserine. “Proteinogenic amino acids” refers to the amino acidswhich occur in natural proteins, i.e., in the proteins ofmicroorganisms, plants, animals and humans, and include L-aspartic acid,L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan, L-arginine, L-proline and, where appropriate,L-seleno-cysteine and L-pyrrolysine. Particularly preferredproteinogenic amino acids are L-lysine, L-isoleucine, L-valine andL-proline. As used herein, the terms “amino acids” or “L-amino acids”includes salts thereof such as, for example, lysine monohydrochloride orlysine sulfate.

L-lysine-producing or -secreting strains of coryneform bacteria include:

-   -   Corynebacterium glutamicum DM58-1/pDM6 (=DSM4697) described in        EP 0 358 940,    -   Corynebacterium glutamicum MH20-22B (=DSM16835) described in        Menkel et al. (Appl. Env. Microbiol. 55(3):684-688 (1989)),    -   Corynebacterium glutamicum AHP-3 (=Ferm BP-7382) described in EP        1 108 790,    -   Corynebacterium glutamicum NRRL B-11474 described in U.S. Pat.        No. 4,275,157, and    -   Corynebacterium thermoaminogenes AJ12521 (═FERM BP-3304)        described in U.S. Pat. No. 5,250,423.

L-valine-producing or -secreting strains of coryneform bacteria include:

Brevibacterium lactofermentum FERM BP-1763 described in U.S. Pat. No.5,188,948,

Brevibacterium lactofermentum FERM BP-3007 described in U.S. Pat. No.5,521,074,

Corynebacterium glutamicum FERM BP-3006 described in U.S. Pat. No.5,521,074, and

Corynebacterium glutamicum FERM BP-1764 described in U.S. Pat. No.5,188,948.

L-isoleucine-producing or -secreting strains of coryneform bacteriainclude:

Brevibacterium flavum FERM BP-355 described in JP 60030693, and

Corynebacterium glutamicum FERM BP-456 described in JP 60030693.

L-proline-producing or -secreting strains of coryneform bacteriainclude:

Brevibacterium lactofermentum NRRL B-11421 described in U.S. Pat. No.4,224,409, and

Corynebacterium glutamicum NRRL B-11423 described in U.S. Pat. No.4,224,409.

L-homoserine-producing or -secreting strains of coryneform bacteriainclude:

Micrococcus glutamicus ATCC 14296 described in U.S. Pat. No. 3,189,526and

Micrococcus glutamicus ATCC 14297 described in U.S. Pat. No. 3,189,526.

Data on the taxonomic classification of strains of these groups ofbacteria may be found, inter alia, in Seiler (J. Gen. Microbiol.129:1433-1477 (1983)), Kinoshita (Glutamic Acid Bacteria, pp. 115-142.In: Demain and Solomon (ed), Biology of Industrial Microorganisms. TheBenjamin/Cummins Publishing Co., London, UK (1985)), Kämpfer et al.(Can. J. Microbiol. 42:989-1005 (1996)), Liebl et al. (Inter. J. System.Bacteriol. 41:255-260 (1991)), Fudou, et al. (Intern. J. System. Evol.Microbiol. 52:1127-1131 (2002)) and in U.S. Pat. No. 5,250,434.

Strains with the designation “ATCC” can be purchased from the AmericanType Culture Collection (Manassas, Va., USA). Strains with thedesignation “DSM” can be purchased from the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ, Brunswick, Germany). Strainswith the designation “NRRL” can be purchased from the AgriculturalResearch Service Patent Culture Collection (ARS, Peoria, Ill., US).Strains with the designation “FERM” can be purchased from NationalInstitute of Advanced Industrial Science and Technology (AIST TsukubaCentral 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan).

In the work on coryneform bacteria which led to the present invention,the sugR gene in the chromosome of these bacteria was identified andcharacterized, and its physiological significance was described (Engels,et al., J. Bacteriol. 189:2955-2966 (2007)). The sugR gene codes for apolypeptide which is referred to as the SugR transcriptional regulator.It is assigned to the DeoR family of transcriptional regulators. Thenucleotide sequence of the sugR gene coding for the SugR regulator ofthe wild type (ATCC13032) of Corynebacterium glutamicum (“wild-typegene”) is depicted in SEQ ID NO:1 and the amino acid sequence, resultingtherefrom is depicted in SEQ ID NO:2 or 4. SEQ ID NO:3 additionallyshows the nucleotide sequences located upstream and downstream of thesugR gene. The terms SugR regulator, SugR transcriptional regulator andSugR polypeptide are mutually interchangeable.

The SugR transcriptional regulator represses, in the presence ofso-called gluconeogenic carbon sources such as, for example, acetic acid(acetate), pyruvic acid (pyruvate) or citric acid (citrate), theexpression of the ptsG, ptsS and ptsF genes. The ptsG gene codes for theglucose-specific component of the phosphotransferase system (PTS)(Handbook of Corynebacterium glutamicum, L. Eggeling and M. Bott (Eds.),CRC Press, 2005). SEQ ID NO:5 depicts the nucleotide sequence of theptsG, including the nucleotide sequences located upstream anddownstream, in Corynebacterium glutamicum ATCC13032. SEQ ID NO:8represents the amino acid sequence of the PtsG polypeptide.

The ptsS gene codes for the sucrose-specific component of thephosphotransferase system and the ptsF gene codes for thefructose-specific component of the phosphotransferase system.

It was found in the work leading to the present invention that the SugRtranscriptional regulator binds to a polynucleotide or a nucleotidesequence (DNA binding motif) which is located upstream of the codingregion of the ptsG gene and includes the nucleotide sequencecorresponding to position 662 to 684 of SEQ ID NO:5.

The sugR gene of coryneform bacteria further includes polynucleotides oralleles which code for variants of the SugR polypeptide which bind withsubstantially the same activity or affinity to a polynucleotide havingthe nucleotide sequence corresponding to position 662 to 684 of SEQ IDNO:5, such as the SugR polypeptide shown in SEQ ID NO:2. Polynucleotidessuitable for such an assay include or possess, for example, thenucleotide sequence from position 618 to 804 of SEQ ID NO:5 or 611 to684 of SEQ ID NO:5.

The activity or affinity of the SugR polypeptide for binding to apolynucleotide can be determined with the aid of retardation gelelectrophoresis. This entails a polypeptide being mixed with apolynucleotide, and the change in mobility of the polypeptide-loadedpolynucleotide fragment in an electric field, typically gelelectrophoresis, is investigated. This design of experiment is alsoknown among those skilled in the art as band shift assay or gelretardation assay.

The term “with substantially the same activity or affinity” means inthis connection that a slowing of the rate of migration of thepolynucleotide loaded with the SugR polypeptide occurs with a 50- to80-fold molar excess, preferably 60- to 80-fold molar excess, andparticularly preferably 65- to 70-fold molar excess, of the SugRpolypeptide based on the polynucleotide employed, which includes thenucleotide sequence corresponding to position 662 to 683 of SEQ ID NO:5.

Variants of the SugR polypeptide are for example those whose amino acidsequence is at least 90%, preferably at least 95%, particularlypreferably at least 98%, and very particularly preferably at least 99%identical to the amino acid sequence of SEQ ID NO:2, with the length ofthe encoded SugR polypeptide preferably comprising 259 amino acids.Examples of such variants are the SugR polypeptide of Corynebacteriumglutamicum R depicted in SEQ ID NO:10 and of Corynebacterium efficiensdepicted in SEQ ID NO:12. The nucleotide sequences of the relevant genesare detailed in SEQ ID NO:9 and 11.

Variants of the SugR polypeptide also include polypeptides comprisingone or more conservative amino acid exchanges relative to SEQ ID NO:2,SEQ ID NO:10 or SEQ ID NO:12. Preferably there should be no more than 10such exchanges, more preferably not more than 5 or 7 such exchanges, andstill more preferably not more than 2 or 3 such exchanges.

In the case of aromatic amino acids, mutual exchanges of phenylalanine,tryptophan and tyrosine are referred to as conservative exchanges. Inthe case of hydrophobic amino acids, mutual exchanges of leucine,isoleucine and valine are referred to as conservative exchanges. In thecase of polar amino acids, mutual exchanges of glutamine and asparagineare referred to as conservative exchanges. In the case of basic aminoacids, mutual exchanges of arginine, lysine and histidine are referredto as conservative exchanges. In the case of acidic amino acids, mutualexchanges of aspartic acid and glutamic acid are referred to asconservative exchanges. In the case of amino acids comprising hydroxylgroups, mutual exchanges of serine and threonine are referred to asconservative exchanges.

Variants of the SugR polypeptide also include polypeptides whichadditionally comprise an extension or truncation of at least one (1)amino acid at the N or C terminus of SEQ ID NO:2, 10 or 12. Thisextension or truncation amounts to not more than 20, 15, 10, 7, 5, 3 or2 amino acids or amino acid residues. Such variants include, inter alia,those in which histidine molecules, for example ten histidines, havebeen attached at the N terminus (histidine tag).

Variants of the SugR polypeptide also include polypeptides in which atleast one (1) amino acid is inserted into or deleted from the amino acidsequence of SEQ ID NO:2, 10 or 12. The maximum number of such changes,referred to as “indels,” may be 2, 3, 4, or 5 but in no case more than 6amino acids.

The term “attenuation” designates, in general, the reduction orelimination of the intracellular activity or concentration of one ormore enzymes or proteins which are encoded by the corresponding DNA in amicroorganism. Attenuation may result from, for example, using a weakerpromoter than in the non-recombinant microorganism or parent strain forthe corresponding enzyme or protein, using a gene or allele which codesfor a corresponding enzyme or protein having a low activity,inactivating the corresponding enzyme or protein or the open readingframe or the gene, and, where appropriate, combining these measures.

“Open reading frame” (ORF) designates a nucleotide sequence segmentwhich may code for a protein, polypeptide or ribonucleic acid, to whichno function can be assigned based upon the prior art. After a functionhas been assigned to the relevant segment of the nucleotide sequence,reference is generally made to a gene. The term “alleles” mean, ingeneral, alternative forms of a given gene. The forms are distinguishedby differences in the nucleotide sequence.

The polypeptide or protein encoded by a gene or an allele, or theencoded ribonucleic acid, is referred to as a “gene product.” The terms“protein” and “polypeptide” are used as synonyms herein. It is knownthat the terminal methionine is deleted during protein synthesis byenzymes intrinsic to the host, called amino peptidases.

A review of known promoters of varying strengths in Corynebacteriumglutamicum may be found in Pátek et al. (J. Biotechnol. 104:311-323(2003)). Further weak promoters are described in communication 512057 ofthe periodical “Research Disclosure” of December 2006 (pages 1616 to1618).

Mutations suitable for attenuating genes include transitions,transversions, insertions and deletions of at least one (1) base pair ornucleotide in the coding region of the relevant gene. Missense mutationslead to an exchange of a given amino acid in a protein fornon-conservative amino acid. As a result, the function or activity ofthe protein is impaired and reduced to a value of from 0 to 75%, 0 to50%, 0 to 25%, 0 to 10% or 0 to 5%. Nonsense mutations lead to a stopcodon in the coding region of the gene and thus to premature ierminationof translation (switching off).

Insertions or deletions of at least one base pair in a gene lead toframe shift mutations which result in incorrect amino acids beingincorporated or translation being prematurely terminated. If themutation results in a stop codon in the coding region, this likewiseleads to premature termination of translation. The measures used togenerate a nonsense mutation are preferably carried out in the5′-terminal part of genes, which codes for the N terminus of thepolypeptide. If the total length of a polypeptide (measured as number ofchemically connected L-amino acids) is designated at 100%, the Nterminus of the polypeptide includes—in the context of the presentinvention—the part of the amino acid sequence which, calculated from theL-formylmethionine starting amino acid, comprises 80% of the subsequentL-amino acids.

In vivo mutagenesis methods are described, for example, in the Manual ofMethods for General Bacteriology (Gerhard et al. (eds.), AmericanSociety for Microbiology, Washington, D.C., USA, 1981) or in Tosaka etal. (Agri. Biol. Chem. 42(4):745-752 (1978)) or in Konicek, et al.(Folia Microbiologica 33:337-343 (1988)). Suitable methods for in vitromutagenesis include treatment with hydroxylamine as disclosed by Miller(Miller, J. H.: A Short Course in Bacterial Genetics. A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 1992), the use ofmutagenic oligonucleotides (Brown: Gentechnologie für Einsteiger,Spektrum Akademischer Verlag, Heidelberg, 1993 and Horton, Mol. Biotech.3:93-99 (1995)) and the use of a polymerase chain reaction employing aDNA polymerase which shows a high error rate. One example of such a DNApolymerase is the Mutazyme DNA polymerase (GeneMorph PCR MutagenesisKit, No. 600550) supplied by Stratagene (LaJolla, Calif., USA).

Further instructions and reviews on the generation of mutations in vivoor in vitro can be found in the prior art and known textbooks ofgenetics and molecular biology such as, for example, the textbook ofKnippers (“Molekulare Genetik”, 6th edition, Georg Thieme Verlag,Stuttgart, Germany, 1995), that of Winnacker (“Gene and Klone”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990) or that of Hagemann(“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986).

The method of gene or allele exchange, which is described in principlein Schwarzer and Pühler (Bio/Technology 9:84-87 (1991)) can be used totransfer a mutation produced in vitro, or a polynucleotide comprisingthe desired mutation, into the chromosome. Schäfer et al. (Gene145:69-73 (1994)) employed this method to incorporate a deletion intothe hom-thrB operon of C. glutamicum. Nakagawa et al. (EP 1108790) andOhnishi et al. (Appl. Microbiol. Biotechnol. 58(2):217-223 (2002))employed this method to incorporate various mutations starting from theisolated alleles into the chromosome of C. glutamicum.

One method for targeted reduction of gene expression consists of placingthe gene to be attenuated under the control of a promoter which can beinduced by addition of metered amounts of IPTG (isopropylβ-D-thiogalactopyranoside), such as, for example, the trc promoter orthe tac promoter. Vectors that may be used for this purpose include, forexample, the Escherichia coli expression vector pXK99E (WO0226787;deposited in accordance with the Budapest Treaty on 31 Jul. 2001 inDH5alpha/pXK99E as DSM14440 at the Deutsche Sammlung für Mikroorganismenand Zellkulturen (DSMZ, Brunswick, Germany)), pEKEx2 (NCBI Accession No.AY585307) or PVWEx2 (Wendisch, Ph. D. thesis, Berichte desForschungszentrums Jülich, Jül-3397, ISSN 0994-2952, Jülich, Germany(1997)), which make IPTG-dependent expression of the cloned genepossible in Corynebacterium glutamicum. This method has been employed,for example, in WO0226787 for the regulated expression of the deaD geneby integration of the vector pXK99EdeaD into the genome ofCorynebacterium glutamicum and by Simic, et al. (Appl. Environ.Microbiol. 68:3321-3327 (2002)) for the regulated expression of the glyAgene by integration of the vector pK18mobglyA′ into Corynebacteriumglutamicum.

A further method for specifically reducing gene expression is theantisense technique, in which short oligodeoxynucleotides or vectors arebrought into target cells to synthesize longer antisense RNA. Theantisense RNA is able to bind there to complementary segments ofspecific mRNAs and reduce their stability, or block translatability. Oneexample thereof is to be found by the skilled person in Srivastava, etal. (Appl. Environ. Microbiol. 66(10):4366-4371 (2000)).

The rate of elongation is influenced by the codon used, and it ispossible to use codons for t-RNAs that occur rarely in the parent strainto attenuate gene expression. It is also possible to exchange an ATGstart codon for a less commonly occurring GTG or TTG codon to impairtranslation. In this regard, it should be noted that the AUG codon istwo to three times more efficient than the GUG and UUG codons (Khudyakovet al., FEBS Letters 232(2):369-71 (1988); Reddy et al., Proc. Nat'lAcad. Sci. USA 82 (17):5656-60 (1985)).

Attenuated forms of the sugR gene or of other genes can be detected withthe aid of 1- and 2-dimensional protein gel fractionation and subsequentvisual identification of the protein concentration in the gel withappropriate evaluation software. A useful method for preparing theprotein gels in the case of coryneform bacteria and for identifying theproteins is the procedure described by Hermann, et al. (Electrophoresis,22:1712-23 (2001)). The protein concentration can likewise be analyzedby Western blot hybridization with an antibody specific for the proteinto be detected (Sambrook, et al., Molecular cloning: a laboratorymanual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989) and subsequent visual evaluation with appropriate softwareto determine concentration (Lohaus et al., Biospektrum 5:32-39 (1998);Lottspeich, Angewandte Chemie 111:2630-2647 (1999)).

The activity of DNA-binding proteins can be measured by means of a DNAband shift assay (also referred to as gel retardation) (Wilson, et al.,J. Bacteriol. 183:2151-2155 (2001)). The effect of DNA-binding proteinson the expression of other genes can be detected by variouswell-described reporter gene assay methods (Sambrook et al., Molecularcloning: a laboratory manual. 2nd Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989). The intracellular enzymaticactivities can be determined by various described methods (Donahue, etal., J. Bacteriol. 182(19):5624-5627 (2000); Ray, et al., J. Bacteriol.182(8):2277-2284 (2000); Treedburg, et al., J. Bacteriol.115(3):816-823) (1973)).

Attenuation measures generally reduce the activity or concentration ofthe corresponding protein to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0to 5% of the activity or concentration of the wild-type protein, or ofthe activity or concentration of the protein in the non-recombinantmicroorganism or parent strain for the corresponding enzyme or protein.“Non-recombinant microorganism” or “parent strain” means themicroorganism on which attenuation measures are carried out.

The invention preferably relates to a recombinant coryneform bacteriumwhich produces an organic chemical compound and in which the sugR genewhich codes for an SugR regulator or SugR polypeptide has beenattenuated. Prior to attenuation, the SugR regulator has an amino acidsequence corresponding to that described above.

The SugR regulator preferably includes or possesses an amino acidsequence from the group consisting of:

-   -   a) amino acid sequence of SEQ ID NO:2, 10 or 12 including one or        more conservative amino acid exchanges, and preferably the amino        acid sequence of SEQ ID NO:2 including one or more conservative        amino acid exchanges and    -   b) amino acid sequence of SEQ ID NO:2, 10 or 12, preferably the        amino acid sequence of SEQ ID NO:2.

The invention preferably relates to coryneform bacteria in which theattenuation of expression of the sugR gene is achieved by one or more ofthe measures selected from the group of

-   -   a) replacement of the nucleobase thymine at position 946 of SEQ        ID NO:3 by guanine,    -   b) deletion of one or more of the nucleobases from position 941        to 946, preferably deletion of all nucleobases from position 941        to 946, of SEQ ID NO:3,    -   c) deletion of one or more of the nucleobases between position        991 and 996 of SEQ ID NO:3,    -   d) replacement of one or more of the nucleobases adenine or        guanine between position 991 and 996 of SEQ ID NO:3 by thymine        or cytosine.    -   e) exchange of the ATG start codon at position 1 to 3 of SEQ ID        NO: 1 for a GTG or TTG.

The promoter region of the sugR gene is shown in SEQ ID NO:13. Position1 of SEQ ID NO: 13 corresponds to position 940 of SEQ ID NO: 3.Positions 2, 7, 52 and 57 of SEQ ID NO:13 are in accordance withpositions 941, 946, 991 and 996 of SEQ ID NO:3. Directly connected tothe 3′ end of the promoter region is the coding region of the sugR gene(or variant of the sugR gene), as for instance shown in SEQ ID NO:3.

The invention preferably relates to coryneform bacteria in which theattenuation of expression of the sugR gene is achieved by one or more ofthe measures selected from the group of

-   -   a) replacement of the nucleobase thymine at position 7 of SEQ ID        NO:13 by guanine,    -   b) deletion of one or more of the nucleobases from position 2 to        7, preferably deletion of all nucleobases from position 2 to 7,        of SEQ ID NO:13,    -   c) deletion of one or more of the nucleobases between position        52 and 57 of SEQ ID NO:13,    -   d) replacement of one or more of the nucleobases adenine or        guanine between position 52 and 57 of SEQ ID NO:13 by thymine or        cytosine.

The invention further relates preferably to coryneform bacteria in whichthe attenuation of the sugR gene is achieved by one or more of themeasures of amino acid exchange selected from the group of

-   -   a) exchange of L-arginine at position 37 of SEQ ID NO:2, 10 or        12, preferably SEQ ID NO:2, for an amino acid selected from the        group of L-alanine, glycine, L-isoleucine and L-proline,        preferably L-proline,    -   b) exchange of L-arginine at position 38 of SEQ ID NO:2, 10 or        12, preferably SEQ ID NO:2, for an amino acid selected from the        group of L-alanine, glycine, L-isoleucine and L-proline,        preferably L-proline,    -   c) exchange of L-aspartic acid at position 39 of SEQ ID NO:2, 10        or 12, preferably SEQ ID NO:2, for an amino acid selected from        the group of L-alanine, glycine, L-isoleucine and L-proline,        preferably L-proline,    -   d) exchange of L-leucine at position 40 of SEQ ID NO:2, 10 or        12, preferably SEQ ID NO:2, for L-proline,    -   e) exchange of L-arginine at position 72 of SEQ ID NO:2, 10 or        12, preferably SEQ ID NO:2, for an amino acid selected from the        group of L-alanine, glycine, L-glutamic acid and L-aspartic        acid, preferably L-alanine,    -   f) exchange of L-aspartic acid at position 101 of SEQ ID NO:2,        10 or 12, preferably SEQ ID NO:2, for an amino acid selected        from the group of L-arginine, L-lysine, L-phenylalanine,        L-methionine, L-glutamine, L-tryptophan, L-tyrosine and        L-glutamic acid, preferably L-arginine,    -   g) exchange of L-threonine at position 105 of SEQ ID NO:2, 10 or        12, preferably SEQ ID NO:2, for an amino acid selected from the        group of L-proline, L-phenylalanine, L-isoleucine, L-methionine,        L-glutamine, L-tryptophan and L-tyrosine, preferably L-proline,    -   h) exchange of L-valine at position 210 of SEQ ID NO:2, 10 or        12, preferably SEQ ID NO:2, for an amino acid selected from the        group of L-alanine, L-arginine, and L-proline, preferably        L-alanine, and    -   i) exchange of L-lysine at position 216 of SEQ ID NO:2, 10 or        12, preferably SEQ ID NO:2, for an amino acid sequence selected        from the group of L-alanine, L-glutamic acid, L-isoleucine and        L-tryptophan, preferably L-alanine.

The stated measures of amino acid exchange in the polypeptide shown inSEQ ID NO:2, 10 or 12, preferably SEQ ID NO:2, can be combined with oneor more measures of deletion or replacement of nucleotides at positions946, 941 to 946, 991 to 996 of SEQ ID NO:3 and positions 1 to 3 of SEQID NO:2, as stated above.

The invention finally relates to coryneform bacteria in which theswitching off of the sugR gene is achieved by a mutagenesis within thecoding region of the sugR gene selected from the group of

-   -   a) insertion of one or more nucleobases,    -   b) deletion of one or more nucleobases,    -   c) deletion of one or more codons, and    -   d) transversion or transition of one or more nucleobase(s) which        leads to at least one stop codon,        where the mutagenesis measure for switching off preferably takes        place within the nucleotide sequence which codes for the amino        acids from position 1 to 230, preferably from position 6 to 58,        of SEQ ID NO:2, 10 or 12. A deletion is a preferred measure,        with particular preference for a deletion of the nucleotide        sequence which codes for the amino acids from position 7 to 248        or 1 to 259 of SEQ ID NO:2, 10 or 12.

L-Lysine-producing coryneform bacteria typically have afeedback-resistant or desensitized aspartate kinase. Feedback-resistantaspartate kinases mean aspartate kinases (LysC) which, in comparisonwith the wild form, exhibit less sensitivity to inhibition by mixturesof lysine and threonine or mixtures of AEC (aminoethylcysteine) andthreonine or lysine alone or AEC alone. The genes or alleles coding forthese desensitized aspartate kinases are also referred to as lysC^(FBR)alleles. Numerous lysC^(FBR) alleles are described in the state of theart and code for aspartate kinase variants which have amino acidexchanges by comparison with the wild-type protein. The coding region ofthe wild-type lysC gene of Corynebacterium glutamicum corresponding tothe access number AX756575 of the NCBI database is depicted in SEQ IDNO:7, and the polypeptide encoded by this gene is depicted in SEQ IDNO:8.

The L-lysine-producing coryneform bacteria used for the inventionpreferably have a lysC allele which codes for a feedback-resistantaspartate kinase. Particularly preferred aspartate kinase variants havethe amino acid sequence of SEQ ID NO:8, the latter including one or moreof the amino acid exchanges selected from the group:

-   -   LysC A279T (L-alanine at position 279 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-threonine; see U.S. Pat. No. 5,688,671 and access numbers        E06825, E06826, E08178 and I74588 to I74597),    -   LysC A279V (L-alanine at position 279 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for L-valine,        see JP 6-261766 and access number E08179),    -   LysC L297Q (L-leucine at position 297 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-glutamine; see DE 102006026328),    -   LysC S301F (L-serine at position 301 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-phenylalanine; see U.S. Pat. No. 6,844,176 and access number        E08180),    -   LysC S301Y (L-serine at position 301 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-tyrosine, see Kalinowski, et al. (Mol. Gen. Genet. 224:317-324        (1990)) and access number X57226),    -   LysC T3081 (L-threonine at position 308 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-isoleucine; see JP 6-261766 and access number E08181)    -   LysC T311I (L-threonine at position 311 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-isoleucine; see WO 00/63388 and U.S. Pat. No. 6,893,848),    -   LysC S317A (L-serine at position 317 of the encoded aspartate        kinase protein according to SEQ ID NO:12 exchanged for        L-alanine; see U.S. Pat. No. 5,688,671 and access number        174589),    -   LysC R320G (L-arginine at position 320 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for glycine;        see Jetten et al. (Appl. Microbiol. Biotechnol. 43:76-82 (1995))        and access number L27125),    -   LysC G345D (glycine at position 345 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for L-aspartic        acid; see Jetten, et al. (Appl. Microbiol. Biotechnol. 43:76-82        (1995)) and access number L16848),    -   LysC T380I (L-threonine at position 380 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-isoleucine; see WO 01/49854 and access number AX192358), and    -   LysC S381F (L-serine at position 381 of the encoded aspartate        kinase protein according to SEQ ID NO:8 exchanged for        L-phenylalanine; see EP 0435132).

The aspartate kinase variants may additionally comprise, whereappropriate, the exchange LysC S317A (L-serine at position 317 of theencoded aspartate kinase protein according to SEQ ID NO:8 exchanged forL-alanine; see U.S. Pat. No. 5,688,671 and access number 174589).

Particular preference is given among the aspartate kinase variants tothe variants LysC T311I (threonine at position 311 of the encodedaspartate kinase protein according to SEQ ID NO:8 exchanged forisoleucine) and the variants comprising at least one exchange selectedfrom the group of A279T (alanine at position 279 of the encodedaspartate kinase protein according to SEQ ID NO:8 exchanged forthreonine), S381F (serine at position 381 of the encoded aspartatekinase protein according to SEQ ID NO:8 exchanged for phenylalanine) andT380I (L-threonine at position 380 of the encoded aspartate kinaseprotein according to SEQ ID NO:8 exchanged for L-isoleucine). The lysCT311I variant (threonine at position 311 of the encoded aspartate kinaseprotein according to SEQ ID NO:8 exchanged for isoleucine) is veryparticularly preferred. The strain DSM 16833 (WO 06/063660) has alysC^(FBR) allele which codes for an aspartate kinase protein whichcomprises the amino acid exchange T311I. The strain NRRL B-11474 (U.S.Pat. No. 4,275,157) has a lysC^(FBR) allele which codes for an aspartatekinase protein which comprises the amino acid exchange S381F. It hasfurther emerged that it is advantageous for lysine production tooverexpress the lysC^(FBR) alleles.

In a further embodiment, the coryneform bacteria employed for themeasures of L-lysine production, which preferably additionally comprisea polynucleotide which codes for a lysine-insensitive aspartate kinasevariant, have one or more of the features selected from the group:

-   -   a) overexpressed polynucleotide (asd gene) which codes for an        aspartate-semialdehyde dehydrogenase (Asd),    -   b) overexpressed polynucleotide (dapA gene) which codes for a        dihydrodipicolinate synthase (DapA),    -   c) overexpressed polynucleotide (dapB gene) which codes for a        dihydropicolinate reductase (DapB),    -   d) overexpressed polynucleotide (dapD gene) which codes for a        tetrahydrodipicolinate succinylase (DapD),    -   e) overexpressed polynucleotide (dapC gene) which codes for a        succinyl-aminoketopimelate transaminase (DapC),    -   f) overexpressed polynucleotide (dapE) which codes for a        succinyl-diaminopimelate desuccinylase (DapE),    -   g) overexpressed polynucleotide (ddh gene) which codes for a        diaminopimelate dehydrogenase (Ddh),    -   h) overexpressed polynucleotide (dapF gene) which codes for a        diaminopimelate epimerase (DapF),    -   i) overexpressed polynucleotide (lysA gene) which codes for a        diaminopimelate decarboxylase (LysA),    -   j) overexpressed polynucleotide (lysE gene) which codes for a        polypeptide having L-lysine export activity (LysE),    -   k) overexpressed polynucleotide (aat gene) which codes for an        aspartate aminotransferase (Aat),    -   1) overexpressed polynucleotide (pyc gene) which codes for a        pyruvate carboxylase (Pyc),    -   m) eliminated or attenuated activity of the malate-quinone        oxidoreductase (Mqo), encoded by the mqo gene,    -   n) eliminated or attenuated activity of the malate dehydrogenase        (Mdh) encoded by the mdh gene,    -   o) eliminated or attenuated activity of the citrate synthase        (GltA) encoded by the gltA gene.

Genes known in the prior art can be used for overexpression of thestated genes or polynucleotides, for example the so-called wild-typegenes of Escherichia coli (Blattner et al., Science 277(5):1453-1462(1997)), Bacillus subtilis (Kunst, et al, Nature 390:249-256 (1977)),Bacillus licheniformis (Veith et al, J. Mol. Microbiol. Biotechnol.7(4):204-211 (2004)), Mycobacterium tuberculosis (Fleischmann et al, J.Bacteriol. 1841:5479-5490 (2004)), Mycobacterium bovis (Garnier et al,Proc. Nat'l Acad. Sci. USA. 100 (13):7877-7882 (2003)), Streptomycescoelicolor (Redenbach, et al, Mol. Microbiol. 21(1):77-96 (1996)),Lactobacillus acidophilus (Alternann et al, Proc. Nat'l Acad. Sci. USA102(11):3906-3912 (2005)), Lactobacillus johnsonii (Pridmore, et al,Proc. Nat'l Acad. Sci. USA 101(8):2512-2517 (2004)), Bifidobacteriumlongum (Schell, et al, Proc. Nat'l Acad. Sci. USA 99(22):14422-14427(2002)), and Saccharomyces cerevisiae. The genomes of the wild-typeforms of these bacteria are available in sequenced and annotated form.The endogenous genes or polynucleotides of the genum Corynebacterium,particularly preferably of the species Corynebacterium glutamicum, arepreferably used.

The nucleic acid sequences can be taken from the databases of theNational Center for Biotechnology Information (NCBI) of the NationalLibrary of Medicine (Bethesda, Md., USA), the nucleotide sequencedatabase of the European Molecular Biologies Laboratories (EMBL,Heidelberg, Germany and Cambridge, UK) or the DNA database of Japan(DDBJ, Mishima, Japan).

Endogenous genes and polynucleotides mean respectively the open readingframes (ORF), genes or alleles, and polynucleotides thereof, present inthe population of a species. The dapA gene of Corynebacterium glutamicumATCC13032 strain is described for example in EP 0 197 335. It isadditionally possible to employ for overexpression of the dapA gene ofCorynebacterium glutamicum, inter alia, the mutations MC20 and MA16 asdescribed in U.S. Pat. No. 6,861,246. EC No. 4.2.1.52 is assigned todihydrodipicolinate synthase activity.

The asd gene of Corynebacterium glutamicum ATCC 21529 strain isdescribed for example in U.S. Pat. No. 6,927,046. EC No. 1.2.1.11 isassigned to aspartate-semialdehyde dehydrogenase activity.

The lysA gene of Corynebacterium glutamicum ATCC13869 (Brevibacteriumlactofermentum) is described for example in U.S. Pat. No. 6,090,597. ECNo. 4.1.1.20 is assigned to diaminopimelate decarboxylase activity.

The aat gene of Corynebacterium glutamicum ATCC13032 is described forexample in Kalinowski, et al (J. Biotechnol. 104(1-3):5-25 (2003); seealso access number NC_(—)006958). It is referred to therein as aspBgene. A gene coding for an aspartate aminotransferase is referred to asaspC in U.S. Pat. No. 6,004,773. Marienhagen, et al (J. Bacteriol.187(22):7693-7646 (2005) refer to the aat gene as aspT gene. EC No.2.6.1.1 is assigned to aspartate aminotransferase activity.

The lysE gene of Corynebacterium glutamicum R127 is described forexample in U.S. Pat. No. 6,858,406. The R127 strain is arestriction-defective mutant of ATCC13032 (Liebl, et al, FEMS Microbiol.Lett. 65:299-304 (1989)). The lysE gene of the ATCC13032 strain used inU.S. Pat. No. 6,861,246 can be employed in the same way.

The pyc gene of Corynebacterium glutamicum of the ATCC 13032 strain isdescribed for example in WO 99/18228 and WO 00/39305. It is furtherpossible to use alleles of the pyc gene as are described for example inU.S. Pat. No. 6,965,021. The pyruvate carboxylases described in thispatent have one or more of the amino acid exchanges selected from thegroup: Pyc E153D (L-glutamic acid at position 153 exchanged forL-aspartic acid), Pyc A182S (L-alanine at position 182 exchanged forL-serine), Pyc A206S (L-alanine at position 206 exchanged for L-serine),Pyc H227R (L-histidine at position 227 exchanged for L-arginine), PycA455G (L-alanine at position 455 exchanged for glycine), and Pyc D1120E(L-aspartic acid at position 1120 exchanged for L-glutamic acid). Thepyc allele which codes for a pyruvate carboxylase which comprises theamino acid exchange Pyc P458S (L-proline at position 458 exchanged forL-serine), and which is described in EP 1 108 790 can be used in thesame way. EC No. 6.4.1.1 is assigned to pyruvate carboxylase.

The ddh gene of Corynebacterium glutamicum ATCC 13869 strain isdescribed for example in U.S. Pat. No. 6,090,597. EC No. 1.4.1.16 isassigned to meso-diaminopimelate dehydrogenase.

Genetic measures for eliminating malate-quinone oxidoreductase (Mqo) aredescribed for example in U.S. Pat. No. 7,094,106. EC No. 1.1.99.16 isassigned to malate-quinone oxidoreductase.

Genetic measures for eliminating malate dehydrogenase (Mdh) aredescribed for example in WO 02/02778. EC No. 1.1.1.37 is assigned tomalate dehydrogenase.

It is likewise possible by suitable amino acid exchanges to reduce thecatalytic property of the relevant polypeptide. In the case ofmalate-quinone oxidoreductase (Mqo) this can be achieved, as describedin WO 06/077004, by producing and using alleles of the mqo gene whichcode for an Mqo variant which has the amino acid sequence as describedin WO 06/077004 and comprises one or more amino acid exchanges selectedfrom the group of

-   -   a) L-serine at position 111 exchanged for another proteinogenic        amino acid, preferably L-phenylalanine or L-alanine, and    -   b) L-alanine at position 201 exchanged for another proteinogenic        amino acid, preferably L-serine.

Particularly preferred strains comprise an mqo allele which codes for anMqo variant which comprises L-phenylalanine at position 111.

In the case of citrate synthase (GltA), a reduction in catalyticproperties can be achieved, as described in PCT/EP2007/056153, byproducing and using alleles of the gltA gene which code for a GltAvariant which has the amino acid sequence as described inPCT/EP2007/056153, where L-aspartic acid at position 5 is replaced byanother proteinogenic amino acid, preferably L-valine, L-leucine andL-isoleucine, particularly preferably L-valine. EC No. 4.1.3.7 isassigned to citrate synthase.

L-Valine-producing coryneform bacteria typically have afeedback-resistant or desensitized acetolactate synthase(acetohydroxyacid synthase; EC No. 2.2.1.6). Feedback-resistantacetolactate synthase means an acetolactate synthase which, bycomparison with the wild form, shows a lower sensitivity to inhibitionby one or more of the amino acids selected from the group of L-valine,L-isoleucine and L-leucine, preferably L-valine.

The acetolactate synthase (IlvB, IlvN) of Corynebacterium consists of aso-called large subunit encoded by the ilvB gene and of a so-calledsmall subunit encoded by the ilvN gene (Keilhauer et al., J. Bacteriol.175(17)5595-5603 (1993)). WO 05/003357 and Elisakova et al. (Appl.Environ. Microbiol. 71(1):207-13 (2005)) report on variants of the IlvNsubunit which confer resistance to L-valine, L-isoleucine and L-leucineon the acetolactate synthase. One variant comprises at position 21 ofthe amino acid sequence L-aspartic acid instead of L-isoleucine (IlvNI21D) and at position 22 L-phenylalanine instead of L-isoleucine (IlvNI22F). The second variant comprises at position 20 of the amino acidsequence L-aspartic acid instead of glycine (IlvN G20D), at position 21of the amino acid sequence L-aspartic acid instead of L-isoleucine (IlvNI21D) and at position 22 L-phenylalanine instead of L-isoleucine (IlvNI22F). It is advantageous where appropriate to overexpress the geneswhich code for the wild form of acetolactate synthase, or the alleleswhich code for a feedback-resistant or desensitized acetolactatesynthase.

The coryneform bacteria used for L-valine production may additionallyhave one or more of the features selected from the group:

-   -   a) overexpressed polynucleotide (ilvC gene) which codes for an        isomeroreductase (IlvC, EC 1.1.1.86),    -   b) overexpressed polynucleotide (ilvD gene) which codes for a        dihydroxy-acid dehydratase (IlvD, EC 4.2.1.9),    -   c) overexpressed polynucleotide (ilvE gene) which codes for a        transaminase B (IlvE, EC 2.6.1.42), and    -   d) eliminated or attenuated activity of the E1p subunit encoded        by the aceE gene of the pyruvate dehydrogenase complex (EC No.        1.2.4.1).

The ilvC gene of C. glutamicum coding for the isomeroreductase has beendescribed for example by Keilhauer, et al. (J. Bacteriol.175(17):5595-603 (1993) and in EP1108790 (see also access numbers C48648and AX127147). The ilvD gene of C. glutamicum coding for thedihydroxy-dehydratase has been described for example in EP1006189 (seealso access number AX136925). The ilvE gene of C. glutamicum coding fortransaminase B (EC 2.6.1.42) is described for example in EP1108790 (seealso access numbers AX127150 and AX122498). Joint overexpression of theilvB, ilvN, ilvC and ilvD genes of Corynebacterium glutamicum can beachieved for example with the aid of the plasmid pECM3ilvBNCD describedin EP 1 006 189. This plasmid is deposited in the form of theEscherichia coli K12 strain DH5αmcr/pECM3ilvBNCD as DSM12457 at theDSMZ. Measures for switching off and attenuating the aceE gene aredescribed in EP 1 767 616.

The term “overexpression” describes in this connection an increase inthe intracellular activity or concentration of one or more enzymes orproteins which are encoded by the corresponding DNA in a microorganismby, for example, increasing the copy number of the gene or genes oralleles or using a strong promoter. Numerous promoters which make itpossible to adjust a desired concentration or activity of thepolypeptide or of the enzyme are described in the prior art. Forexample, the lysC promoter of the mutant DM58-1, which is described inKalinowski et al. (Mol. Microbiol. 5(5):1197-1204 (1991), or the gappromoter, which is described in the patent application with theapplication number EP 06007373.1, can be employed. It is furthermorepossible to employ the promoters described in the patent applicationwith the application number EP 06117294.6, the promoters described byPatek, et al. (J. Biotechnol. 104(1-3):311-323 (2003), or the variantsof the dapA promoter, for example the A25 promoter, described byVasicova, et al. (J. Bacteriol. 181:6188-6191 (1999)).

It is also possible to employ promoters known from the genetics ofEscherichia coli, such as, for example, tac promoter, trp promoter, trcpromoter and lpp promoter or the P_(L) and P_(R) promoter of phage λ.

The gene or polynucleotide which has been provided with a promoter insuch a way can be incorporated in the form of one (1) or more copiesinto the desired coryneform bacterium by using methods of transformationor conjugation as are sufficiently well known in the prior art, andwhere appropriate also by ballistic methods. It is possible to use forthis purpose for example plasmids which are replicated by coryneformbacteria. A large number of such plasmids are described in the priorart. Suitable plasmid vectors are for example pZ1 (Menkel et al., Appl.Environ. Microbiol. 64:549-554 (1989) or the pSELF vectors described byTauch et al. (J. Biotechnol. 99:79-91 (2002)). A review article on thetopic of plasmids in Corynebacterium glutamicum is to be found in Tauch,et al. (J. Biotechnol. 104:27-40 (2003)).

A further possibility is to introduce one or more copies of the relevantgene, typically a maximum of 20, preferably a maximum of 10 to a maximumof 5, into the chromosome of a coryneform bacterium (geneamplification). In one embodiment, using for example methods describedin Reinscheid et al. (Appl. Environ. Microbiol. 60:126-132 (1994)) forthe hom-thrB operon, a plasmid which is non-replicative in C. glutamicumand which comprises the gene of interest is transferred into acoryneform bacterium. After homologous recombination by means of acrossover event, the resulting strain comprises at least two copies ofthe relevant gene.

In another embodiment, using for example methods described in WO03/040373 and US-2003-0219881-A1, one or more copy (copies) of the geneof interest is introduced into a desired site in the chromosome of C.glutamicum by means of at least two recombination events.

In a further embodiment, using for example, methods described in WO03/014330 and US-2004-0043458-A1, a tandem duplication of the gene canbe achieved.

Finally, it is possible to adjust the copy number of the gene with theaid of transposons or IS elements (see: U.S. Pat. No. 5,804,414 or U.S.Pat. No. 5,591,577).

The concentration of overexpressed protein can be determined by 1- and2-dimensional protein gel fractionation and subsequent visualidentification of the protein concentration in the gel using appropriateevaluation software. A suitable method for preparing the protein gels inthe case of coryneform bacteria and for identifying the proteins is theprocedure described by Hermann, et al. (Electrophoresis 22:1712-23(2001)). The protein concentration can likewise be determined by Westernblot hybridization with an antibody specific for the protein to bedetected (Sambrook, et al., Molecular cloning: a laboratory manual. 2ndEd. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)and subsequent visual evaluation with appropriate software to determinethe concentration (Lohaus, et al., Biospektrum 5:32-39 (1998);Lottspeich, Angewandte Chemie 38: 2630-2647 (1999)). It is likewisepossible to use the enzyme assay described in the prior art to determinethe activity.

Overexpression measures may increase the activity or concentration ofthe corresponding protein by at least 10%, 25%, 75%, 100%, 150%, 200%,300%, 400% or 500%, maximally up to 1000% or 2000% based on that of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

In the work leading to the present invention it was found thatcoryneform bacteria in which the sugR gene is present in attenuatedform, when cultured in a medium which comprises a carbon source whichconsists substantially of one or more of the sugars selected from thegroup of glucose, fructose and sucrose, and of one or more organic acidsselected from the group of acetic acid, citric acid and pyruvic acid,preferably acetic acid, and/or alcohols, preferably glycerol, produceorganic chemical compounds with an increased product formation ratecompared with coryneform bacteria in which the sugR gene is present inthe form of the wild-type gene or in which the sugR gene has not beenattenuated.

The term “product formation rate” describes the increase in the productconcentration per unit time. A useful unit is g/l·h (grams per litre andhour). The term specific product formation rate takes account of theconcentration of the bacteria or of the biomass of bacteria at the givenperiod or time. A useful unit is g(product)/g(biomass)·h(grams(product)per gram(biomass)and hour).

The invention accordingly also relates to a process for the fermentativepreparation of organic chemical compounds, characterized in that itcomprises the following steps:

-   -   a) culturing or fermentation of the recombinant coryneform        bacteria which produce the desired organic chemical compound and        in which the sugR gene is present in attenuated form, using a        nutrient medium, and    -   b) under conditions with which the desired organic chemical        compound is enriched or accumulated in the medium and/or in the        cells, where the nutrient medium comprises a carbon source which        substantially consists of one or more of the sugars selected        from the group of glucose, fructose and sucrose, and one or more        of the organic acids selected from the group of acetic acid,        citric acid, pyruvic acid, preferably acetic acid, and/or        alcohols, preferably glycerol, and where appropriate,    -   c) obtaining or isolating the desired organic chemical compound,        with where appropriate further constituents of the fermentation        broth and/or the biomass remain in their totality or in portions        (>0 to 100%) in the final product,        where the concentration of the organic chemical compound is        measured where appropriate at one or more different times during        the progress of the process. The bacteria which can be employed        in step a) of the process include inter alia the coryneform        bacteria described herein.

The produced microorganisms are cultured according to the invention in abatch process (batch cultivation), in a fed-batch process, in a repeatedfed-batch process or in a continuous process. Summaries about suchprocesses are available in the textbook by Chmiel (Bioprozesstechnik 1.Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag,Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren andperiphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

In a batch process, with few exceptions such as, for example, oxygen andpH-correcting means, all the starting materials are introduced in theform of a batch, and the microorganism is cultured in the resultingmedium.

In a fed-batch process, the microorganism is initially cultured by meansof a batch process (batch phase). Subsequently, a starting materialwhich is essential for preparation of the product, where appropriatealso a plurality of starting materials, is added continuously ordiscontinuously (feed phase). In the case of the preparation accordingto the invention of an organic chemical compound, this is a carbonsource.

A repeated fed-batch process is a fed-batch process in which, aftercompletion of the fermentation, part of the resulting fermentation brothis used as inoculum to start a renewed repeated fed-batch process. Thiscycle can be repeated where appropriate more than once.

Repeated fed-batch processes are described for example in WO 02/18543and WO 05/014843.

A continuous process entails a batch or fed-batch process being followedby continuous addition of one or more, where appropriate all, startingmaterials to the culture and, at the same time, removal of fermentationbroth. Continuous processes are described for example in the patentsU.S. Pat. No. 5,763,230, WO 05/014840, WO 05/014841 and WO 05/014842.

The culture medium or fermentation medium to be used must satisfy in asuitable manner the demands of the respective strains. The fermentationmedium or the media employed during the fermentation comprise(s) all thesubstances or components ensuring growth of the microorganism andformation of the desired organic chemical compound. Descriptions ofculture media of various microorganisms are to be found in the handbook“Manual of Methods for General Bacteriology” of the American Society ofBacteriology (Washington D.C., USA, 1981). The terms culture medium,fermentation medium and nutrient medium or medium are mutuallyexchangeable.

Descriptions of fermentation media are present inter alia in U.S. Pat.No. 6,221,636, in U.S. Pat. No. 5,840,551, in U.S. Pat. No. 5,770,409,in U.S. Pat. No. 5,605,818, in U.S. Pat. No. 5,275,940, in U.S. Pat. No.4,275,157 and in U.S. Pat. No. 4,224,409. A culture medium generallycomprises inter alia one or more carbon source(s), nitrogen source(s)and phosphorus source(s). The culture medium must additionally comprisesalts of metals such as, for example, magnesium sulfate or iron sulfate,which are necessary for growth. Finally, essential growth factors suchas amino acids and vitamins may be employed in addition to theabovementioned substances.

Carbon source means in the context of this invention compounds whichcomprise only carbon, oxygen and hydrogen in the molecule and areutilized by the microorganisms to form their biomass and to produce anorganic chemical compound. The carbon source in the process of theinvention consists essentially of one or more of the sugars selectedfrom the group of glucose, fructose and sucrose, and one or more of theorganic acids selected from the group of acetic acid, citric acid andpyruvic acid. In the case of organic acids, acetic acid is preferred.Mixtures of sugars and organic acids may result inter alia on hydrolysisof lignocellulose.

A preferred process is one in which the carbon source consistsessentially of a mixture of one or more of the sugars selected from thegroup of glucose, fructose and sucrose, and acetic acid. Preference isgiven to glucose, sucrose and sugar mixtures essentially consisting ofglucose and fructose, or sugar mixtures essentially consisting ofsucrose, glucose and fructose. Very particular preference is given toglucose- and sucrose-containing mixtures.

The term “essentially” takes account of the fact that not onlychemically pure starting materials but also, where appropriate, impurestarting materials of technical or lower quality are used in industrialfermentation processes to prepare organic chemical compounds. The termfurther takes account of the composition of typical complexmicrobiological media constituents, the chemical change in the startingmaterials as a result of sterilization by heat, and finally thetransfer, resulting through the inoculation of a nutrient medium bymeans of a preculture (an inoculum), of media constituents orconstituents of the fermentation broth.

The term “acetic acid” includes not only the acid itself but also thesalts of acetic acid such as the ammonium salt, the alkali metal salts,for example the potassium salt, or the alkaline earth metal salts, forexample the calcium salt. It is possible to use in a process of theinvention not only chemically pure acetic acid but also acetic acid oftechnical quality. Acetic acid of technical quality comprises lowconcentrations of compounds which arise in the preparation process andhave not been completely removed.

Starch hydrolyzates are an important source of glucose. Accordingly,concomitant substances are typically for example maltose and/orisomaltose in concentrations of approximately 0.1 to 2% by weight. Theglucose used for the measures of the invention has a content of ≧(atleast) 90% by weight, preferably ≧95% by weight (based on dry matter).

It is known that the sucrose in sucrose solutions is converted toglucose and fructose to a small extent at elevated temperature like thatpresent for example during heat sterilization. The content of glucoseand fructose in the sucrose or sucrose solution employed for themeasures of the invention is ≦(not more than) 10% by weight, preferably≦5% by weight (based on dry matter). The molasses resulting from theproduction of sucrose from sugarbeet (beet molasses) comprises a sugarmixture which consists essentially of sucrose.

Concentrated solutions comprising glucose and fructose are known toskilled persons for example under the designation “isosyrup”, “highfructose corn syrup” or “fructose-containing glucose syrup”. These areprepared by hydrolysis of starch with subsequent treatment of theglucose with isomerase. They comprise a sugar mixture which generallycomprises approximately 50-55% by weight glucose and 40-45% by weightfructose (based on dry matter). Owing to the preparation process, suchsyrups generally comprise inter alia maltose in a concentration notexceeding approximately 6% by weight (based on dry matter).

Concentrated solutions essentially comprising a sugar mixture consistingof equimolar portions of glucose and fructose are likewise known toskilled persons. These solutions are prepared for example by treatingsucrose with invertase and therefore generally comprise small amounts ofsucrose (≦5% by weight based on dry matter). Concentrated solutionscomprising a sugar mixture consisting of sucrose, glucose and fructoseare further known to skilled persons. These solutions are prepared bypartial inversion of sucrose. One known concentrate consists of 38.5% byweight sucrose, 38.5% by weight invert sugar (i.e., glucose and fructosein the ratio 1:1) and 23% by weight water. The molasses resulting fromthe production of sucrose from sugar cane (sugar cane molasses)comprises a sugar mixture which consists essentially of sucrose, glucoseand fructose.

The sugar mixtures described above are suitable as carbon source for theprocess of the invention.

The term “essentially” further takes account of the fact that complexmedia constituents employed in the fermentation medium, such as, forexample, yeast extract or corn steep liquor, comprise varyingproportions of compounds able to serve as carbon source. Thus, yeastextract comprises inter alia trehalose and/or glucose. Corn steep liquorcomprises approximately 10 to 20% by weight (based on dry matter) lacticacid. Such complex media constituents are generally employed ifnecessary in a concentration of 0.5 to 20 g/l and also in higherconcentrations in the fermentation medium.

Information on the production of glucose, sucrose and fructose and onthe composition of complex media constituents are to be found inter aliain the textbooks by Bartens (Zuckertechnologie, Rüben andRohrzuckerherstellung, Verlag Dr. Albert Bartens K G, Berlin (Germany)),McGinnis (Beet-Sugar Technology, third edition, Studio of Printcraft,Fort Collins, Colo., US (1982)), Drews (Mikrogiologisches Praktikum, 3rdedition, Springer Verlag, Berlin (Germany), 1976), Rehm (IndustrielleMikrobiologie, 2nd edition, Springer Verlag, Berlin (Germany), 1980) andCrueger and Crueger (Lehrbuch der Angewandten Mikrobiologie, AkademischeVerlagsgesellschaft, Wiesbaden (Germany), 1982).

The term “essentially” finally takes account the fact that inoculationof a nutrient medium by using a so-called preculture (inoculum)transfers carbon sources which have not been consumed or have beenformed during the preculture into the culture used to prepare therespective organic chemical compound. The proportion of the preculturein the culture resulting from the inoculation generally amounts to from2 to 20%, where appropriate also not more than 50%.

The acetic acid employed in a process of the invention generallyconsists of ≧(at least) 90% by weight, preferably at least 92.5% byweight and particularly preferably ≧95% by weight of acetic acid (basedon the anhydrous starting material). Acetic acid with a purity of ≧97%by weight, ≧98% by weight or ≧99% by weight can likewise be employed.

The sugar preferably employed, or the sugar mixtures preferablyemployed, in a process of the invention generally consist(s) of ≧(atleast) 90% by weight, where appropriate preferably ≧95% by weight, ofone or more of the stated sugars selected from the group of glucose,sucrose and fructose (based on dry matter).

If mixtures consisting of acetic acid and glucose, or mixturesconsisting of acetic acid and sucrose, are employed in a process of theinvention, the proportion of acetic acid in the mixture is ≧(at least)5% by weight, ≧10% by weight, ≧20% by weight, ≧40% by weight, ≧50% byweight, ≧60% by weight, or ≧80% by weight and ≦(at most)≦90% by weightor ≦(at most) 95% by weight.

If mixtures consisting of acetic acid and of a sugar mixture composed ofglucose and fructose are employed in a process of the invention, theproportion of acetic acid in the mixture is ≧(at least) 5% by weight,≧10% by weight, ≧20% by weight, ≧40% by weight, ≧50% by weight, ≧60% byweight, or ≧80% by weight and ≦(at most) 90% by weight. The proportionof glucose in the sugar mixture consisting of glucose and fructose is≧(at least) 5% by weight, ≧10% by weight, ≧20% by weight, ≧40% byweight, ≧50% by weight, ≧60% by weight, or ≧80% by weight and ≦(at most)90% by weight, preferably 45 to 65% by weight, 50 to 60% by weight, 50to 60% by weight, 55 to 65% by weight or 55 to 60% by weight.

If mixtures consisting of acetic acid and of a sugar mixture composed ofsucrose, glucose and fructose are employed in a process of theinvention, the proportion of acetic acid in the mixture is ≧(at least)5% by weight, ≧10% by weight, ≧20% by weight, ≧40% by weight, ≧50% byweight, ≧60% by weight, or ≧80% by weight and ≦(at most) 90% by weightor ≦(at most) 95% by weight. The proportion of sucrose, glucose andfructose in the sugar mixture is in each case >(more than) 10% by weightand less than 80% by weight.

The total amount of the individual components in the mixture of thecarbon sources adds up to 100% by weight. The carbon sources arenaturally present in the fermentation media in the form of aqueoussolutions.

The concentration of acetic acid during the fermentation generally doesnot exceed 40 g/l or 30 g/l. The concentration of acetic acid during thefermentation is where appropriate not more than 20 g/l, 10 g/l, 5 g/l or2.5 g/l. If acetic acid and one or more of the sugars selected from thegroup of glucose, sucrose and fructose is employed as carbon source in aprocess of the invention, the medium or the fermentation broth in thecase of the batch process accordingly then comprises at least at thestart of the process acetic acid and the stated sugar(s) as carbonsource.

In the case of a fed-batch process, the carbon source, i.e. said sugarsand the acetic acid, can be introduced into the process in various ways.In a first aspect, one or more of the stated sugars is introduced forthe batch phase. In the subsequent feed phase,

-   -   a) acetic acid, or    -   b) a mixture of acetic acid and one or more of the stated sugars        is employed. The feed phase preferably starts before the        sugar(s) introduced at the start of the batch phase has (have)        been completely consumed.

In a second aspect, acetic acid is introduced for the batch phase. Inthe subsequent feed phase,

-   -   a) a mixture of acetic acid and one or more of the stated sugars        is employed, or    -   b) a mixture of one or more of the stated sugars is employed.

The feed phase preferably starts before the acetic acid introduced atthe start of the batch phase has been completely consumed.

In a third aspect, a mixture of acetic acid and one or more of thestated sugars is introduced for the batch phase. In the subsequent feedphase,

-   -   a) acetic acid, or    -   b) a mixture of acetic acid and one or more of the stated sugars        is employed.

In a fourth aspect, a carbon source selected from the group of: a)acetic acid; b) one or more of the stated sugars; and c) a mixture ofacetic acid and one or more of the sugars selected from the group ofsucrose, glucose and fructose; is introduced for the batch phase. In thesubsequent feed phase: d) acetic acid is employed in at least onecontinuous period of at least one (1) hour and not more than 100,preferably not more than 50, particularly preferably not more than 30hours, and subsequently one or more of the stated sugars is employed inat least one continuous period of at least one (1) hour and not morethan 100, preferably not more than 50, particularly preferably not morethan 30 hours; or e) one or more of the stated sugars is employed in atleast one continuous period of at least one (1) hour and not more than100, preferably not more than 50, particularly preferably not more than30 hours, and subsequently acetic acid is employed in at least onecontinuous period of at least one (1) hour and not more than 100,preferably not more than 50, particularly preferably not more than 30hours, where the proportion of acetic acid in the carbon source employedis ≧(at least) 5% by weight, ≧10% by weight, ≧20% by weight, ≧40% byweight, ≧50% by weight, ≧60% by weight, or ≧80% by weight and ≦(at most)90% by weight or ≦(at most) 95% by weight. The feed phase preferablystarts before the carbon source(s) introduced at the start of the batchphase has (have) been completely consumed. The term “completelyconsumed” means that the respective carbon source is no longerdetectable in the fermentation broth.

In the fed-batch processes, the third aspect mentioned with case b) ispreferred.

In the case of a repeated fed-batch process, the carbon sources employedfor repeated batch and feed phases are as described for the batchprocess and fed-batch process (see above).

In the case of a continuous culture, acetic acid and one or more of thesugars selected from the group of sucrose, glucose and fructose areadded continuously.

It is possible to employ as a nitrogen source inorganic compounds suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate, preferably ammonium sulfate, and organiccompounds such as urea. The nitrogen sources can be used singly or asmixture. Additionally employed where appropriate are organicnitrogen-containing substance mixtures such as peptones, yeast extract,meat extract, corn steep liquor or soya flour.

It is possible to use as phosphorus source phosphoric acid, potassiumdihydrogenphosphate, dipotassium hydrogenphosphate or the correspondingsodium-containing salts, singly or as mixture.

Fermentations is generally carried out at a pH of from 5.5 to 9.0, inparticular 6.0 to 8.0. To control the pH of the culture, basic compoundssuch as sodium hydroxide, potassium hydroxide, ammonia or aqueousammonia or acidic compounds such as phosphoric acid or sulfuric acid areemployed in a suitable manner. To control foaming it is possible toemploy antifoams such as, for example, fatty acid polyglycol esters. Tomaintain the stability of plasmids it is possible to add suitableselectively acting substances, for example antibiotics, to the medium.In order to maintain aerobic conditions, oxygen or oxygen-containing gasmixtures such as, for example, air are introduced into the culture.

The temperature of the culture is normally 25° C. to 45° C. andpreferably 30° C. to 40° C. The activity of the microorganisms resultsin an enrichment or accumulation of the organic chemical compound in thefermentation or culture broth. The culture is continued until formationof the desired organic chemical compound is maximal. This aim isnormally achieved within 10 hours to 160 hours. Longer culturing timesare possible in continuous processes.

A “fermentation broth” or “culture broth” refers to a fermentationmedium in which a microorganism has been cultured for a certain time andat a certain temperature. When the fermentation is complete, theresulting fermentation broth accordingly comprises a) the biomassproduced as a result of the growth of the cells of the microorganism, b)the organic chemical compound formed during the fermentation, c) theorganic by-products formed during the fermentation, and d) theconstituents of the fermentation medium/media employed or of thestarting materials such as, for example, vitamins such as thiamine orsalts such as magnesium sulfate, which have not been consumed by thefermentation.

The produced culture or fermentation broth is then collected and thedesired organic chemical compound present in the fermentation broth, andthe product comprising the desired organic chemical compound areobtained or isolated. A solid or liquid product is then obtained byfurther processing steps. In this way, the desired organic chemicalcompound present in the fermentation broth is converted into a desiredproduct formed.

Methods for determining organic chemical compounds are known in theprior art. Analysis of, for example, L-amino acids can take place asdescribed by Spackman, et al. (Anal. Chem. 30:1190 (1958)) by anionexchange chromatography with subsequent ninhydrin derivatization, or itcan take place by reversed phase HPLC as described by Lindroth, et al.(Anal. Chem. 51:1167-1174 (1979)).

In the case of the amino acid L-lysine, substantially four differentproduct forms are known in the state of the art. One group ofL-lysine-containing products includes concentrated aqueous alkalinesolutions of purified L-lysine (EP-B-0534865). A further group asdescribed for example in U.S. Pat. No. 6,340,486 and U.S. Pat. No.6,465,025 includes aqueous acidic biomass-containing concentrates ofL-lysine-containing fermentation broths. The best-known group of solidproducts includes powder or crystalline forms of purified or pureL-lysine which is typically in the form of a salt such as, for example,L-lysine monohydrochloride. A further group of solid product forms isdescribed for example in EP-B-0533039. Besides L-lysine, the productform described therein comprises most of the starting materials whichwere used during the fermentative production and were not consumed and,where appropriate, the biomass of the microorganism employed with acontent of >0%-100%.

In the case of the amino acids L-valine, L-isoleucine, L-proline,L-tryptophan and L-homoserine, the product forms known in the prior artare substantially those containing the relevant amino acids in purifiedor pure form (>95% by weight or >98% by weight).

Corresponding to the different product forms, a wide variety ofprocesses are known with which the L-amino acid is collected, isolatedor purified from the fermentation broth in order to produce the L-aminoacid-containing product or the purified L-amino acid.

Solid pure L-amino acids are produced substantially by using methods ofion exchange chromatography, where appropriate with use of activatedcarbon, and methods of crystallization. In the case of lysine, thecorresponding base or a corresponding salt such as, for example, themonohydrochloride (Lys-HCl) or lysine sulfate (Lys₂-H₂SO₄) is obtainedin this way.

In the case of lysine, EP-B-0534865 describes a process for producingaqueous, basic L-lysine-containing solutions from fermentation broths.In the process described therein, the biomass is removed from thefermentation broth and discarded. A base such as, for example, sodium,potassium or ammonium hydroxide is used to adjust a pH of between 9 to11. The mineral constituents (inorganic salts) are removed from thebroth after concentration and cooling by crystallization and either usedas fertilizers or discarded.

In processes for producing lysine using the bacteria according to theinvention, processes resulting in products which comprise components ofthe fermentation broth are also employed. These are used in particularas animal feed additives.

Biomass can be removed wholly or partly from the fermentation broth byseparation methods such as, for example, centrifugation, filtration,decantation or a combination thereof, or be left completely therein.Where appropriate, the biomass or the biomass-containing fermentationbroth is inactivated during a suitable process step, for example bythermal treatment (heating) or by addition of acid. The chemicalconstituents of the biomass are, inter alia, the cell envelope, forexample the peptidoglycan and the arabinogalactan, the protein orpolypeptide, lipids and phospholipids and nucleic acids (DNA and RNA).

In one procedure, the biomass is removed completely or almost completelyso that no (0%) or not more than 30%, not more than 20%, not more than10%, not more than 5%, not more than 1% or not more than 0.1% biomassremains in the product produced. In a further procedure, the biomass isnot removed, or is removed only in small proportions, so that all (100%)or more than 70%, 80%, 90%, 95%, 99% or 99.9% biomass remains in theproduct produced. In one process according to the invention,accordingly, the biomass is removed in proportions ≧ to 0% to ≦100%.

Finally, the fermentation broth obtained after the fermentation can beadjusted, before or after the complete or partial removal of thebiomass, to an acidic pH with an inorganic acid such as, for example,hydrochloric acid, sulfuric acid or phosphoric acid or organic acid suchas, for example, propionic acid (GB 1,439,728 or EP 1 331 220). It islikewise possible to acidify the fermentation broth with the completecontent of biomass (U.S. Pat. No. 6,340,486 or U.S. Pat. No. 6,465,025).Finally, the broth can also be stabilized by adding sodium bisulfite(NaHSO₃, GB 1,439,728) or another salt, for example ammonium, alkalimetal or alkaline earth metal salt of sulfurous acid.

During the removal of the biomass, organic or inorganic solids presentwhere appropriate in the fermentation broth are partially or completelyremoved. The organic by-products dissolved in the fermentation broth andthe dissolved unconsumed components of the fermentation medium (startingmaterials) remain at least partly (>0%), preferably to the extent of atleast 25%, particularly preferably to the extent of at least 50% andvery particularly preferably to the extent of at least 75% in theproduct. Where appropriate, they also remain completely (100%) or almostcompletely, meaning >95% or >98%, in the product. In this sense, theterm “based on fermentation broth” means that the product comprises atleast part of the components of the fermentation broth. in addition ofthe desired organic chemical compound.

Subsequently, water is removed from the broth by known methods such as,for example, with the aid of a rotary evaporator, thin-film evaporator,falling film evaporator, by reverse osmosis or by nanofiltration. Thisconcentrated fermentation broth can then be worked up to free-flowingproducts, in particular to a fine-particle powder or preferably coarsegranules, by methods of freeze drying, of spray drying, of spraygranulation or by other processes as described for example in thecirculating fluidized bed according to PCT/EP2004/006655. A desiredproduct is isolated where appropriate from the resulting granules byscreening or dust removal. It is likewise possible to dry thefermentation broth directly, i.e. without previous concentration byspray drying or spray granulation.

“Free-flowing” means powders which flow unimpeded out of a series ofglass orifice vessels with orifices of different sizes at least out ofthe vessel with a 5 mm (millimeters) orifice (Klein: Seifen, Öle, Fette,Wachse 94, 12 (1968)).

“Fine-particle” means a powder predominantly (>50%) of a particle sizeof diameter from 20 to 200 μm.

“Coarse” means a product predominantly (>50%) of a particle size ofdiameter from 200 to 2000 μm.

The particle size determination can be carried out by methods of laserdiffraction spectrometry. Corresponding methods are described in thetextbook on “Teilchengröβenmessung in der Laborpraxis” by R. H. Müllerand R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996)or in the textbook “Introduction to Particle Technology” by M. Rhodes,published by Wiley & Sons (1998).

The free-flowing, fine-particle powder can in turn be converted bysuitable compaction or granulation processes into a coarse, veryfree-flowing, storable and substantially dust-free product.

The term “dust-free” means that the product comprises only smallproportions (<5%) of particle sizes below 100 μm in diameter.

“Storable” in the sense of this invention means a product which can bestored for at least one (1) year or longer, preferably at least 1.5years or longer, particularly preferably two (2) years or longer, in adry and cool environment without any substantial loss (<5%) of therespective amino acid occurring.

The invention accordingly further relates to a process for producing anL-amino acid, preferably L-lysine or L-tryptophan, containing product,preferably animal feed additive, from fermentation broths, characterizedby the steps

-   -   a) culturing and fermentation of an L-amino acid-secreting        coryneform bacterium in which the sugR gene is present in        attenuated form, in a fermentation medium, and accumulation of        the L-amino acid,    -   b) removal of the biomass formed during the fermentation in an        amount of from 0 to 100% by weight, and    -   c) drying of the fermentation broth obtained as in a) and/or b)        in order to obtain the product in the desired powder or granular        form,    -   where an acid selected from the group of sulfuric acid,        phosphoric acid or hydrochloric acid is added where appropriate        before step b) or c). Step a) or b) is preferably followed by        removal of water from the L-amino acid-containing fermentation        broth (concentration). In addition, the concentration of the        L-amino acid is measured where appropriate at one or more of the        process stages.

The invention further relates to a process for producing a lysinesulfate-containing product which is described in principle in DE102006016158, and in which the fermentation broth obtained using themicroorganisms according to the invention, from which the biomass hasbeen removed completely or partly where appropriate, is furtherprocessed by carrying out a process which includes at least thefollowing steps:

-   -   a) the pH is reduced by adding sulfuric acid to 4.0 to 5.2, in        particular 4.9 to 5.1, and a molar sulfate/L-lysine ratio of        from 0.85 to 1.2, preferably 0.9 to 1.0, particularly        preferably >0.9 to <0.95, is adjusted in the broth, where        appropriate by adding a further or a plurality of        sulfate-containing compound(s) and    -   b) the mixture obtained in this way is concentrated by removal        of water, and granulated where appropriate,    -   where one or both of the following measures is/are carried out        where appropriate before step a):    -   c) measurement of the molar sulfate/L-lysine ratio to ascertain        the required amount of sulfate-containing compound(s)    -   d) addition of a sulfate-containing compound selected from the        group of ammonium sulfate, ammonium bisulfate and sulfuric acid        in appropriate ratios.

Where appropriate, also before step b), a salt of sulfurous acid,preferably alkali metal bisulfite, particularly preferably sodiumbisulfite, is added in a concentration of 0.01 to 0.5 by weight,preferably 0.1 to 0.3% by weight, particularly preferably 0.1 to 0.2% byweight, based on the fermentation broth.

Preferred sulfate-containing compounds which should be mentioned in thecontext of the abovementioned process steps are in particular ammoniumsulfate and/or ammonium bisulfate or corresponding mixtures of ammoniaand sulfuric acid and sulfuric acid itself.

The molar sulfate/L-lysine ratio V is calculated by the formula:V=2×[SO₄ ²⁻]/[L-lysine]. This formula takes account of the fact that theSO₄ ²⁻ anion has two charges. A ratio of V=1 means that thestoichiometric composition Lys₂(SO₄) is present, whereas the findingwith a ratio of V=0.9 is a 10% sulfate deficit and with a ratio of V=1.1is a 10% sulfate excess.

It is advantageous to employ during the granulation or compaction theusual organic or inorganic auxiliaries or carriers such as starch,gelatin, cellulose derivatives or similar substances, as normally usedin the processing of food products or feeds as binders, gelling agentsor thickeners, or further substances such as, for example, silicas,silicates (EP0743016A) or stearates.

It is further advantageous to provide the surface of the resultinggranules with oils as described in WO 04/054381. Oils which can be usedare mineral oils, vegetable oils or mixtures of vegetable oils. Examplesof such oils are soya oil, olive oil, soya oil/lecithin mixtures. In thesame way, silicone oils, polyethylene glycols or hydroxyethylcelluloseare also suitable. Treatment of the surfaces with the said oils achievesan increased abrasion resistance of the product and a reduction in thedust content. The oil content in the product is 0.02 to 2.0% by weight,preferably 0.02 to 1.0% by weight, and very particularly preferably 0.2to 1.0% by weight based on the total amount of the feed additive.

Preferred products have a proportion of ≧97% by weight of a particlesize of from 100 to 1800 μm or a proportion of ≧95% by weight of aparticle size of from 300 to 1800 μm diameter. The proportion of dust,i.e. particles with a particle size <100 μm, is preferably >0 to 1% byweight, particularly preferably not exceeding 0.5% by weight.

However, alternatively, the product may also be adsorbed on an organicor inorganic carrier known and customary in the processing of feeds,such as, for example, silicas, silicates, meals, brans, flours,starches, sugars or others, and/or be mixed and stabilized withcustomary thickeners and binders. Examples of use and processes thereforare described in the literature (Die Mühle+Mischfuttertechnik 132 (1995)49, page 817).

Finally, the product can also be brought by coating processes withfilm-formers such as, for example, metal carbonates, silicas, silicates,alginates, stearates, starches, gums and cellulose ethers as describedin DE-C-4100920 to a state in which it is stable to digestion by animalstomachs, especially the stomach of ruminants.

To adjust a desired amino acid concentration in the product it ispossible, depending on requirements, to add the appropriate amino acidduring the process in the form of a concentrate or, if appropriate, of asubstantially pure substance or its salt in liquid or solid form. Thesecan be added singly or as mixtures to the resulting or concentratedfermentation broth, or else during the drying or granulation process.

The invention further relates to a process for producing a solidlysine-containing product as described in principle in US 20050220933,and which includes the working up of the fermentation broth obtainedusing the microorganisms according to the invention, in the followingsteps:

-   -   a) filtration of the fermentation broth, preferably with a        membrane filter, to result in a biomass-containing sludge and a        filtrate,    -   b) concentration of the filtrate, preferably so as to result in        a solids content of from 48 to 52% by weight,    -   c) granulation of the concentrate obtained in step b),        preferably at a temperature of from 50° C. to 62° C., and    -   d) coating of the granules obtained in c) with one or more of        the coating agent(s).

The coating agents used for the coating in step d) are preferablyselected from the group consisting of

-   -   d1) the biomass obtained in step a),    -   d2) an L-lysine-containing compound, preferably selected from        the group of L-lysine hydrochloride or L-lysine sulfate,    -   d3) a substantially L-lysine-free substance with an L-lysine        content of <1% by weight, preferably <0.5% by weight, preferably        selected from the group of starch, carageenan, agar, silicas,        silicates, meals, brans and flours, and    -   d4) a water-repellent substance, preferably selected from the        group of oils, polyethylene glycols and liquid paraffins.

In the case of lysine, the ratio of the ions during the production oflysine-containing products is preferably adjusted so that the equivalention ratio corresponding to the following formula 2x[SO₄ ²⁻]+[Cl⁻]−[NH₄⁺]−[Na⁺]−[K⁺]−2x[Mg²⁺]−2x[Ca²⁺]/[L-Lys] results in 0.68 to 0.95,preferably 0.68 to 0.90, as described by Kushiki et al. in US20030152633 (the molar concentrations are to be given in the “[ ]”).

In the case of lysine, the solid product produced in this way has, basedon the fermentation broth, a lysine content (as lysine base) of 10% byweight to 70% by weight or 20% by weight to 70% by weight, preferably30% by weight to 70% by weight and very particularly preferably of 40%by weight to 70% by weight, based on the dry matter of the product.Maximum contents of lysine base of 71% by weight, 72% by weight, 73% byweight are likewise possible.

The water content of the solid product is up to 5% by weight, preferablyup to 4% by weight, and particularly preferably less than 3% by weight.

The invention therefore relates to an L-lysine-containing feed additivebased on fermentation broth, which exhibits the following features

-   -   a) a lysine content (as base) of at least 10% by weight up to a        maximum of 73% by weight,    -   b) a water content not exceeding 5% by weight, and    -   c) a biomass content corresponding to at least 0.1% of the        biomass present in the fermentation broth, where the biomass,        inactivated where appropriate, is formed by coryneform bacteria        according to the invention.

The present invention is explained in more detail below on the basis ofexemplary embodiments.

EXAMPLES Example 1 Deletion of the sugR Gene

Chromosomal DNA was isolated from the strain ATCC 13032 by the method ofEikmanns et al. (Microbiol. 140:1817-1828 (1994)). On the basis of theknown C. glutamicum sugR gene sequence from the database of the NationalCenter for Biotechnology Information (NCBI) of the National Library ofMedicine (Bethesda, Md., USA), under Accession Number NC_(—)003450(Region: 2037815-2038594, synonym NCg11856) and Accession NumberNC_(—)006958 (Region: 2007864-2008643, synonym cg2115), theoligonucleotides described below were selected for generation of thesugR deletion allele by means of the polymerase chain reaction (PCR)according to the “Splicing by Overlap Extension” method (Gene SOEingmethod) (Horton, Molecular Biotechnology 3: 93-98 (1995)):

Primer sugR_A (SEQ ID NO: 14): 5′- GC GAATTC ACA AGG ATT CATCTG GCA TC -3′ Primer sugR_B (SEQ ID NO: 15):5′- CCCATCCACTAAACTTAAACA GCG CTC CTC TGC GTA CAT -3′Primer sugR_C (SEQ ID NO: 16): 5′- TGTTTAAGTTTAGTGGATGGG CGAGAA CGC GAT GTA GAA GTT GTG -3′ Primer sugR_D (SEQ ID NO: 17):5′- GC GGATCC CAA ATT GCC ACC CAA CAA CAC CC -3′

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany)and the PCR reaction was carried out using Pfu polymerase (Stratagene,Product NO: 600135, La Jolla, USA).

The primers sugR_B and sugR_C are composed of two regions, a sequence ofnucleotides which binds to the nucleotides 1 to 18 and 745 to 768,respectively, within the coding sequence of sugR (see SEQ ID NO:1), anda linker of 21 bp in length. The sequences of the sugR_A and sugR_Dprimers are modified so as to produce recognition sites for restrictionenzymes. The EcoRI recognition sequence is chosen for the sugR_A primerand the BamHI recognition sequence is chosen for the sugR_D primer, bothof which are indicated by underlining in the sequence of nucleotidesdepicted above.

With the aid of the polymerase chain reaction, the sugR_A and sugR_Bprimers enable a 464 bp DNA fragment to be amplified and the sugR_C andsugR_D primers enable a 472 bp DNA fragment to be amplified. Theamplicons are verified by electrophoresis in a 0.8% strength agarosegel, isolated from said agarose gel using the High Pure PCR ProductPurification Kit (Product NO: 1732676, Roche Diagnostics GmbH, Mannheim,Germany) and used together as template for another PCR reaction usingthe sugR_A and sugR_D primers. In this way the 915 bp sugR deletionderivative is generated (see also SEQ ID NO: 18).

The product amplified in this way is verified by electrophoresis in a0.8% strength agarose gel.

Example 2 Cloning of the sugR Deletion Derivative into the pGEM-T Vector

The 915 bp amplified DNA fragment carrying the sugR deletion derivativewas ligated by T4 DNA ligase (pGem-T Vector System, Promega, Wis., USA)into the pGEM-T vector (Promega, Wis., USA).

Subsequently, the E. coli strain DH5α (Grant, et al., Proc. Nat'l Acad.Sci. USA 87:4645-4649 (1990)) is transformed with the ligation mixtureaccording to Hanahan (Techniques for transformation of E. coli, p.109-135 (1985). In G.D.M. (ed.), DNA cloning, vol. 1, IRL-Press,Oxford/Washington D.C.). Plasmid-carrying cells are selected by platingout the transformation mixture on LB agar (Sambrook et al., MolecularCloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) supplemented with 25 mg/lkanamycin. Plasmid DNA is isolated from a transformant with the aid ofthe QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany) and checkedby treatment with the restriction enzymes BamHI and EcoRI or BglI andsubsequent agarose gel electrophoresis (0.8%). The plasmid is referredto as pGEM-T_delsugR. The sugR deletion derivative was verified bysequencing (Agowa GmbH, Berlin, Germany). The sequence is depicted inSEQ ID NO: 18.

Example 3 Construction of the Exchange Vector pK19mobsacB_deltasugR

The sugR deletion derivative is isolated from the plasmid described inExample 2, pGEM-T_delsugR, by complete cleavage with the enzymes EcoRIand BamHI. After fractionation in an agarose gel (0.8%), the approx. 0.9kb fragment carrying the sugR deletion derivative is isolated from saidagarose gel using the High Pure PCR Product Purification Kit (ProductNO: 1732676, Roche Diagnostics GmbH, Mannheim, Germany).

The sugR deletion derivative obtained in this way is used for ligationwith the mobilizable cloning vector pK19mobsacB (Schäfer, et al., Gene14:69-73 (1994)). The latter has been cleaved completely beforehand,using the restriction endonucleases EcoRI and BamHI. The vector preparedin this way is mixed with the sugR deletion allele and treated with T4DNA ligase (Amersham Pharmacia, Freiburg, Germany).

The E. coli strain DH5α (Grant, Proc. Nat'l Acad. Sci. USA 87:4645-4649(1990)) is then electrophorated with the ligation mixture (Hanahan, In.DNA Cloning. A Practical Approach, Vol. 1, ILR-Press, Cold SpringHarbor, N.Y., 1989). The plasmid-carrying cells are selected by platingout the transformation mixture on LB agar (Sambrook et al., MolecularCloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, N.Y., 1989)supplemented with 25 mg/l kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprepSpin Miniprep Kit from Qiagen, and the cloned sugR deletion allele isverified by means of restriction cleavage with the restrictionendonucleases EcoRI and BamHI or PvuII. The plasmid is referred to aspK19mobsacB_deltasugR and is depicted in FIG. 1. The strain is referredto as E. coliH5α/pK19mobsacB_deltasugR.

Example 4 Deletion Mutagenesis of the SugR Gene in C. glutamicum DM1729

The Corynebacterium glutamicum strain DM1729 is a mutant ofCorynebacterium glutamicum ATCC13032, which carries the alleles pycP458S, hom V59A and lysC T311I and has been deposited under the nameDSM17576 with the Deutsche Sammlung für Mikroorganismen and Zellkulturen[German Collection of Microorganisms and Cell Cultures] (DSMZ,Braunschweig, Germany) on Sep. 16, 2005.

The vector specified in Example 3, pK19mobsacB_deltasugR, waselectrophorated by the electrophoration method of van der Rest, et al.(Appl. Microbiol. Biotechnol. 52:541-545 (1999)) into theCorynebacterium glutamicum strain DM1729 (conditions: 25 μF, 600 S2 and2.5 kV/cm (Bio-Rad Gene Pulser Xcell, Bio-Rad Laboratories, Hercules,Canada)).

The vector cannot self-replicate in DM1729 and is retained in the cellonly if it has integrated into the chromosome as a result of arecombination event. Clones with integrated pK19mobsacB_deltasugR areselected by plating out the conjugation mixture on LB agar (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, N.Y., 1989) supplemented with 15 mg/l kanamycin and 50 mg/mlnalidixic acid. Established clones are streaked out on LB agar platescontaining 25 mg/l kanamycin and incubated at 33° C. for 16 hours.Mutants in which the plasmid has been excized as a result of a secondrecombination event are selected by culturing the clones unselectivelyin liquid LB medium for 20 hours, then streaking them out on LB agarcontaining 10% sucrose and incubating them for 24 hours.

Like the starting plasmid, pK19mobsacB, the pK19mobsacB_deltasugRplasmid contains, in addition to the kanamycin resistance gene, a copyof the sacB gene coding for the Bacillus subtilis levan sucrase.Sucrose-inducible expression produces levan sucrase which catalyses thesynthesis of the product levan which is toxic to C. glutamicum. As aresult, only those clones grow on LB agar containing sucrose, in whichthe integrated pK19mobsacB_deltasugR has been excized again. Excisionmay comprise excision of either the complete chromosomal copy of thesugR gene or of the incomplete copy containing the internal deletion,together with the plasmid.

Approximately 40 to 50 colonies are tested for the phenotype “growth inthe presence of sucrose” and “non-growth in the presence of kanamycin”.In order to prove that the deleted sugR allele has remained in thechromosome, 8 colonies having the phenotype “growth in the presence ofsucrose” and “non-growth in the presence of kanamycin” are studied bythe standard PCR method of Innis et al. (PCR Protocols. A Guide toMethods and Applications, 1990, Academic Press) with the aid of thepolymerase chain reaction. In the process, a DNA fragment is amplifiedfrom the chromosomal DNA of the colonies, which carries the sugR geneand surrounding regions. The following primer oligonucleotides areselected for PCR.

sugR-k-for (SEQ ID NO: 19): 5′ GTT CGT CGC GGC AAT GAT TGA CG 3′sugR-k-rev (SEQ ID NO: 20): 5′ CTC ACC ACA TCC ACA AAC CAC GC 3′

In the case of control clones with complete sugR allele, the primersenable an approx. 1.7 kb DNA fragment to be amplified. In the case ofclones having a deleted sugR allele, DNA fragments of approx. 0.96 kbare amplified.

The amplified DNA fragments are identified by means of electrophoresisin a 0.8% strength agarose gel. In this way, 6 of the strain DM1729clones assayed were shown to carry a deleted sugR allele on theirchromosome. One of the clones was referred to as C. glutamicumDM1729deltasugR.

Example 5 Preparation of Lysine

The C. glutamicum strain obtained in Example 4, DM1729deltasugR, iscultured in a nutrient medium suitable for production of lysine, and thelysine content in the culture supernatant is determined.

For this purpose, the strain is first incubated on an agar plate at 33°C. for 24 hours. Starting from this agar plate culture, a preculture isinoculated (10 ml of medium in a 100 ml conical flask). The medium MM isused for said preculture. The preculture is incubated on a shaker at 240rpm and 33° C. for 24 hours. A main culture is inoculated from thispreculture so as to obtain an initial OD (660 nm) of the main culture of0.5 OD. The medium MM is also used for the main culture.

Medium MM CSL 5 g/l MOPS 20 g/l Glucose (autoclaved separately) 50 g/lSalts: (NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7H₂O 1.0 g/l CaCl₂ *2H₂O 10 mg/l FeSO₄ * 7H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/lL-Homoserine (sterile-filtered) 0.4 g/l CaCO₃ 25 g/l

CSL (Corn Steep Liquor), MOPS (Morpholinopropanesulphonic acid) and thesalt solution are adjusted to pH 7 with aqueous ammonia and autoclaved.The sterile substrate and vitamin solutions and also the CaCO₃,autoclaved in the dry state, are added thereafter.

Culturing is carried out in a volume of 10 ml in a 100 ml conical flaskwith baffles. Culturing is carried out at 33° C. and 80% humidity.

After 72 hours, the OD at a measurement wavelength of 600 nm isdetermined using an Ultrospec 3000 (Amersham Pharmacia Biotech GmbH,Freiburg). The amount of lysine produced is determined using an aminoacid analyser from Eppendorf-BioTronik (Hamburg, Germany) by ionexchange chromatography and post-column derivatization with ninhydrindetection. Table 1 depicts the result of the experiment.

TABLE 1 OD Lysine Strain (600 nm) g/l DM1729 18.1 4.9 DM1729deltasugR9.9 5.8

Example 6 Deletion Mutagenesis of the sugR Gene in ATCC13032deltaaceE_deltapqo

The valine-producing Corynebacterium glutamicum strain ATCC13032deltaaceE_deltapqo is a mutant of Corynebacterium glutamicum ATCC13032(Schreiner, et al., J. Bacteriol. 188(4):1341-1350 (2006)).

The sugR gene was deleted as described in Example 4. The strain obtainedwas referred to as C. glutamicum ATCC13032deltaaceE_deltapqo_deltasugR.

C. glutamicum ATCC13032deltaaceE_deltapqo and C. glutamicum ATCC13032deltaaceE_deltapqo_deltasugR were transformed with the plasmidpJC4ilvBNCE (Radmacher et al., Applied and Environmental Microbiology68: 2246-2250 (2002)) according to Van der Rest, et al. (Appl.Microbiol. Biotechnol. 52:541-545 (1999)).

Example 7 Preparation of Valine

The C. glutamicum strain obtained in Example 6,ATCC13032deltaaceE_deltapqo deltasugR/pJC4ilvBNCE, is cultured in anutrient medium suitable for production of valine, and the valinecontent in the culture supernatant is determined.

Comparative fermentations were carried out using the strains C.glutamicum ATCC13032deltaaceE_deltapqo/pJC4ilvBNCE and C.glutamicumATCC13032delta aceE_deltapqo_deltasugR/pJC4ilvBNCE accordingto Blombach, et al. (Appl. Environ. Microbiol. 73(7):2079-2084 (2007)).

C. glutamicum ATCC13032deltaaceE_deltapqo/pJC4ilvBNCE and C. glutamicumATCC13032deltaaceE_deltapqo_deltasugR/pJC4ilvBNCE were drawn byremoving, using a sterile loop, cell suspension from a glycerol cultureand streaking out said cell suspension on a tryptone yeast agar plate(tryptone 16 g/l; yeast extract 10 g/l; NaCl 5 g/l; agar 15 g/l)containing kanamycin (50 μg/ml) and additionally 0.5% (w/v) potassiumacetate (KAc).

After incubating the agar plates at 30° C. for 48 hours, a colony wasused for inoculating 5 ml of tryptone yeast medium (tryptone 16 g/l;yeast extract 10 g/l; NaCl 5 g/l) supplemented with 0.5% (w/v) KAc andkanamycin (50 μg/ml). After 6 h of incubation, the whole 5 ml weretransferred to 50 ml of medium of the same composition and incubatedovernight. For valine fermentation, the cells were pelleted bycentrifugation (using a Centrifuge 5804 R, Eppendorf-Netheler-Hinz GmbH,Cologne, Germany at 5000 rpm; 4° C.; 10 min), washed with 20 ml of 0.9%(w/v) NaCl and added as inoculum to CgXII medium (Keilhauer, et al., J.Bacteriol. 175:5595-5603 (1993)) supplemented with 4% (w/v) glucose, 1%(w/v) KAc, 0.5% (w/v) brain-heart broth and kanamycin (50 μg/ml). C.glutamicum was cultured under aerobic conditions (500 ml conical flaskwith 2 baffles) on a rotary shaker at 120 rpm and 30° C.

The optical density (OD), and the substrate and product concentrationswere determined after 11 hours (according to Blombach et al., Appl.Environ. Microbiol. 73(7):2079-2084 (2007)). Culture growth wasdetermined photometrically (Ultrospec 3000, Amersham Pharmacia BiotechGmbH, Freiburg, Germany) on the basis of the optical density at awavelength of 600 nm. An aliquot of 1 ml of cell suspension wascentrifuged (in a bench centrifuge, type 5415D, (Eppendorf, Hamburg) at13 000 rpm, 10 min, room temperature), and the supernatant was used fordetermining the substrate and product concentrations. The valineconcentration was determined by means of reversed-phase high pressureliquid chromatography (HP 1100, Hewlett-Packard, Waldbronn, Germany)using a fluorescence detector after automated pre-column derivatizationwith ortho-phthaldialdehyde (OPA) (Lindroth, et al, Anal. Chem.51:1667-1674 (1979)). Glucose and acetate concentrations were determinedby enzymatic assays (Roche Diagnostics, Penzberg, Germany).

TABLE 2 Optical density (OD), substrate and product concentrations ofthe fermentation of C. glutamicumATCC13032deltaaceE_deltapqo/pJC4ilvBNCE. C. glutamicumATCC13032deltaaceE_deltapqo/pJC4ilvBNCE Time OD Glucose Acetate L-Valine[h] [600 nm] [g/l] [g/l] [mM] 0 1.02 35.2 12.5 0 11 54.0 27.3 0 1

TABLE 3 Optical density (OD), substrate and product concentrations ofthe fermentation of C. glutamicumATCC13032deltaaceE_deltapqo_deltasugR/pJC4ilvBNCE. C. glutamicumATCC13032deltaaceE_deltapqo_deltasugR/pJC4ilvBNCE Time OD GlucoseAcetate L-Valine [h] [600 nm] [g/l] [g/l] [mM] 0 1.38 44.7 11.3 0 1119.2 35 1.5 10.7

ABBREVIATIONS

The base pair numbers stated are approximate values obtained in thecontext of reproducibility of measurements. The abbreviations anddesignations used have the following meaning:

oriV: ColEl-like origin from pMB1sacB The sacB gene coding for the protein levan sucraseRP4-mob: RP4-mobilization siteKan: Kanamycin resistance geneLacZ-alpha′: 5′ terminus of the lacZα gene fragment'LacZ-alpha: 3′ terminus of the lacZα gene fragmentsugR: Deleted allele of the C. glutamicum sugR geneBamHI: Cleavage site of the restriction enzyme BamHIEcoRI: Cleavage site of the restriction enzyme EcoRIPvuII: Cleavage site of the restriction enzyme PvuII

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by those ofskill in the art that the invention may be practiced within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

1-30. (canceled)
 31. A process for the fermentative production of anorganic chemical compound, comprising: a) producing a fermentation brothby fermenting a recombinant coryneform bacterium in a medium comprisinga carbon source selected from the group consisting of: glucose,fructose, sucrose, and acetic acid, wherein said recombinant coryneformbacterium: i) secretes said organic chemical compound; ii) has theability to utilize as a carbon source one or more of the sugars selectedfrom the group consisting of: glucose, fructose; sucrose, and aceticacid; iii) comprises a feedback resistant aspartate kinase; and iv)comprises a SugR regulator that comprises the sequence of SEQ ID NO:2except for one or more differences selected from the group consistingof: aa) the amino acid at position 37 is selected from the groupconsisting of: L-alanine, glycine, L-isoleucine and L-proline; bb) theamino acid at position 38 is selected from the group consisting of:L-alanine, glycine, L-isoleucine and L-proline; cc) the amino acid atposition 39 is selected from the group consisting of: L-alanine,glycine, L-isoleucine and L-proline; dd) the amino acid at position 40is L-proline; ee) the amino acid at position 72 is selected from thegroup consisting of: L-alanine, glycine, L-glutamic acid and L-asparticacid; ff) the amino acid at position 101 is selected from the groupconsisting of: L-arginine, L-lysine, L-phenylalanine, L-methionine,L-glutamine, L-tryptophan, L-tyrosine and L-glutamic acid; gg) the aminoacid at position 105 is selected from the group consisting of:L-proline, L-phenylalanine, L-isoleucine, L-methionine, L-glutamine,L-tryptophan and L-tyrosine; hh) the amino acid at position 210 isselected from the group consisting of: L-alanine, L-arginine, andL-proline; and ii) the amino acid at position 216 is selected from thegroup consisting of: L-alanine, L-glutamic acid, L-isoleucine andL-tryptophan. b) collecting said fermentation broth containing saidorganic chemical compound.
 32. The process of claim 31, wherein thefermentation broth collected in step b) is dried to form a product basedon the fermentation broth.
 33. The process of claim 31, wherein thefermentation broth collected in step b) is dried without the removal ofbiomass.
 34. The process of claim 31, wherein biomass is removed fromthe fermentation broth of step b) so that not more than 30% of thebiomass remains in the product thereby produced.
 35. The process ofclaim 31, wherein said organic chemical compound is isolated from thefermentation broth of step b) so that not more than 0.1% of biomassremains in the product thereby produced.
 36. The process of claim 31,wherein said organic chemical compound is a hydroxy acid or a keto acid.37. The process of claim 31, wherein said organic chemical compound isan L-amino acid.
 38. The process of claim 31, wherein said organicchemical compound is L-lysine, L-valine or L-isoleucine.
 39. The processof claim 31, wherein said recombinant coryneform bacterium is of thespecies Corynebacterium glutamicum.
 40. The process of claim 31, whereinsaid recombinant coryneform bacterium additionally possesses one or moreof the features selected from the group consisting of: a) overexpressionof a polynucleotide which codes for an aspartate-semialdehydedehydrogenase (Asd), b) overexpression of a polynucleotide which codesfor a dihydrodipicolinate synthase (DapA), c) overexpression of apolynucleotide which codes for a dihydropicolinate reductase (DapB), d)overexpression of a polynucleotide which codes for atetrahydrodipicolinate succinylase (DapD), e) overexpression of apolynucleotide which codes for a succinyl-aminoketopimelate transaminase(DapC), f) overexpression of a polynucleotide which codes for asuccinyl-diaminopimelate desuccinylase (DapE), g) overexpression of apolynucleotide which codes for a diaminopimelate dehydrogenase (Ddh), h)overexpression of a polynucleotide which codes for a diaminopimelateepimerase (DapF), i) overexpression of a polynucleotide which codes fora diaminopimelate decarboxylase (LysA), j) overexpression of apolynucleotide which codes for a polypeptide having L-lysine exportactivity (LysE), k) overexpression of a polynucleotide which codes foran aspartate aminotransferase (Aat), l) overexpression of apolynucleotide which codes for a pyruvate carboxylase (Pyc), m)elimination or attenuation malate-quinone oxidoreductase (Mqo) activity,n) elimination or attenuation of malate dehydrogenase (Mdh) activity,and o) elimination or attenuation of citrate synthase (GltA) activity.41. A process for the fermentative production of an organic chemicalcompound, comprising: a) producing a fermentation broth by fermenting arecombinant coryneform bacterium in a medium comprising a carbon sourceselected from the group consisting of: glucose, fructose, sucrose, andacetic acid, wherein said recombinant coryneform bacterium: i) secretessaid organic chemical compound; ii) has the ability to utilize as acarbon source one or more of the sugars selected from the groupconsisting of: glucose, fructose; sucrose, and acetic acid; iii)comprises a feedback resistant aspartate kinase; and iv) comprises agene encoding the SugR regulator of SEQ ID NO:2, said gene comprising inits promoter region, the nucleotide sequence of SEQ ID NO:13 except forone or more mutations selected from the group consisting of: aa)replacement of the nucleobase thymine at position 7 of SEQ ID NO:13 byguanine; bb) deletion of one or more of the nucleobases from position 2to 7 of SEQ ID NO:13; cc) deletion of one or more of the nucleobasesbetween position 52 and 57 of SEQ ID NO:13; and dd) replacement of oneor more of the nucleobases adenine or guanine between position 52 and 57of SEQ ID NO:13 by thymine or cytosine; b) collecting said fermentationbroth containing said organic chemical compound.
 42. The process ofclaim 41, wherein the fermentation broth collected in step b) is driedto form a product based on the fermentation broth.
 43. The process ofclaim 41, wherein the fermentation broth collected in step b) is driedwithout the removal of biomass.
 44. The process of claim 41, whereinbiomass is removed from the fermentation broth of step b) so that notmore than 30% of the biomass remains in the product thereby produced.45. The process of claim 41, wherein said organic chemical compound isisolated from the fermentation broth of step b) so that not more than0.1% of biomass remains in the product thereby produced.
 46. The processof claim 41, wherein said gene encoding the SugR regulator of SEQ IDNO:2 comprises a start codon selected from the group consisting of GTGand TTG.
 47. The process of claim 41, wherein said organic chemicalcompound is a hydroxy acid or a keto acid.
 48. The process of claim 41,wherein said organic chemical compound is an L-amino acid.
 49. Theprocess of claim 41, wherein said organic chemical compound is L-lysine,L-valine or L-isoleucine.
 50. The process of claim 41, wherein saidrecombinant coryneform bacterium is of the species Corynebacteriumglutamicum.